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Carlo Augusto BORTOLOTTI
Professore Associato
Dipartimento di Scienze della Vita sede ex Chimica V.Campi 103
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Pubblicazioni
2024
- EFFICIENT ELECTROCATALYTIC H2 PRODUCTION BY IMMOBILIZED Co(III)-MYOGLOBIN
[Articolo su rivista]
Meglioli, Mirco; DI ROCCO, Giulia; Ranieri, Antonio; Bortolotti, Carlo Augusto; Sola, Marco; Battistuzzi, Gianantonio; Borsari, Marco
abstract
The thermodynamics and kinetics of heterogeneous electron transfer (ET) for Co-substituted horse myoglobin (Co-Mb) and its derivatives with ammonia and imidazole as heme axial ligands were studied with cyclic voltammetry on a pyrolytic graphite electrode along with their ability to mediate the electrocatalytic production of H2 . All the proteins experience a non-diffusive electrochemical regime as electrode-bound species. The adsorbed Co-Mb construct was found to carry out the electrocatalytic reduction of water protons to H2 with a good efficiency under anaerobic conditions
thus yielding a simple and tunable system for H2 production. Replacement of H2O as Co axial ligand by ammonia and imidazole significantly lowers the catalytic currents for H3O+/H2O reduction to H2.
The E°’ values of the Co(III)/Co(II) redox couple for all species are mainly determined by the enthalpic contribution. Differences were found in the kinetics of ET for the different protein adducts due to changes in the activation enthalpies. However, all species share the same distance of about 14 Å from the electrode surface to the Co(III)/Co(II) center determined using the Marcus model, consistent with a non-denaturing adsorption of the protein.
2024
- How Biorecognition Affects the Electronic Properties of Reduced Graphene Oxide in Electrolyte‐Gated Transistor Immunosensors
[Articolo su rivista]
Sensi, Matteo; de Oliveira, Rafael Furlan; Berto, Marcello; Paradisi, Alessandro; Greco, Pierpaolo; Bortolotti, Carlo Augusto; Samorì, Paolo; Biscarini, Fabio
abstract
Ambipolar electrolyte-gated transistors (EGTs) based on reduced graphene oxide (rGO) have been demonstrated as ultra-sensitive and highly specific immunosensors. However, the physics and chemistry ruling the device operation are still not fully unraveled. In this work, the aim is to elucidate the nature of the observed sensitivity of the device. Toward this aim, a physical–chemical model that, coupled with the experimental characterization of the rGO-EGT, allows one to quantitatively correlate the biorecognition events at the gate electrode and the electronic properties of rGO-EGT is proposed. The equilibrium of biorecognition occurring at the gate electrode is shown to determine the apparent charge neutrality point (CNP) of the rGO channel. The multiparametric analysis of the experimental transfer characteristics of rGO-EGT reveals that the recognition events modulate the CNP voltage, the excess carrier density Δn, and the quantum capacitance of rGO. This analysis also explains why hole and electron carrier mobilities, interfacial capacitance, the curvature of the transfer curve, and the transconductances are insensitive to the target concentration. The understanding of the mechanisms underlying the transistor transduction of the biorecognition events is key for the interpretation of the response of the rGO-EGT immunosensors and to guide the design of novel and more sensitive devices.
2023
- Effects of removal of the axial methionine heme ligand on the binding of S. cerevisiae iso-1 cytochrome c to cardiolipin.
[Articolo su rivista]
Paradisi, Alessandro; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Battistuzzi, Gianantonio
abstract
The cleavage of the axial S(Met)-Fe bond in cytochrome c (cytc) upon binding to
cardiolipin (CL), a glycerophospholipid of the inner mitochondrial membrane, is one of
the key molecular changes that impart cytc with (lipo)peroxidase activity essential to its
pro-apoptotic function. In this work, UV-VIS, CD, MCD and fluorescence
spectroscopies were used to address the role of the Fe−M80 bond in controlling the
cytc-CL interaction, by studying the binding of the Met80Ala (M80A) variant of S.
cerevisiae iso-1 cytc (ycc) to CL liposomes in comparison with the wt protein [Paradisi
et al. J. Biol. Inorg. Chem. 25 (2020) 467–487]. The results show that the integrity of
the six-coordinate heme center along with the distal heme site containing the Met80
ligand is a not requisite for cytc binding to CL. Indeed, deletion of the Fe-S(Met80)
bond has a little impact on the mechanism of ycc-CL interaction, although it results in
an increased heme accessibility to solvent and a reduced structural stability of the
protein. In particular, M80A features a slightly tighter binding to CL at low CL/cytc ratios
compared to wt ycc, possibly due to the lift of some constraints to the insertion of the
CL acyl chains into the protein hydrophobic core. M80A binding to CL maintains the
dependence on the CL-to-cytc mixing scheme displayed by the wt species
2023
- Giving new life to seafood waste: in silico engineering of a chitinolytic LPMO enzyme
[Relazione in Atti di Convegno]
Ricci, A.; Di Rocco, G.; Bortolotti, C. A.; Gautieri, A.
abstract
2023
- Hydrogen Peroxide Induces Heme Degradation and Protein Aggregation in Human Neuroglobin: Roles of the Disulfide Bridge and the H-bonding in the Distal Heme Cavity
[Articolo su rivista]
Di Rocco, G.; Bernini, F.; Battistuzzi, G.; Ranieri, A.; Bortolotti, C. A.; Borsari, M.; Sola, M.
abstract
In this study, human neuroglobin (hNgb) was found to undergo H2O2-induced breakdown of the heme center at a much slower rate than other globins, namely in the timescale of hours against minutes. We studied how the rate of the process is affected by the Cys46/Cys55 disulfide bond and the network of noncovalent interactions in the distal heme side involving Tyr44, Lys67, the His64 heme iron axial ligand and the heme propionate-7. The rate is increased by the Tyr44 to Ala and Phe mutations, however the rate is lowered by Lys67 to Ala swapping. The absence of the disulfide bridge slows down the reaction further. Therefore, the disulfide bond-controlled accessibility of the heme site and the residues at position 44 and 67 affect the activation barrier of the reaction. Wild-type and mutated species form -amyloid aggregates in the presence of H2O2 producing globular structures. Furthermore, the C46A/C55A, Y44A, Y44F and Y44F/C46A/C55A variants yield potentially harmful fibrils. Finally, the nucleation and growth kinetics for the aggregation of the amyloid structures can be successfully described by the Finke-Watzky model.
2023
- Reduced Graphene Oxide Electrolyte-Gated Transistor Immunosensor with Highly Selective Multiparametric Detection of Anti-Drug Antibodies
[Articolo su rivista]
Sensi, M.; de Oliveira, R. F.; Berto, M.; Palmieri, M.; Ruini, E.; Livio, P. A.; Conti, A.; Pinti, M.; Salvarani, C.; Cossarizza, A.; Cabot, J. M.; Ricart, J.; Casalini, S.; Gonzalez-Garcia, M. B.; Fanjul-Bolado, P.; Bortolotti, C. A.; Samori, P.; Biscarini, F.
abstract
The advent of immunotherapies with biological drugs has revolutionized the treatment of cancers and auto-immune diseases. However, in some patients, the production of anti-drug antibodies (ADAs) hampers the drug efficacy. The concentration of ADAs is typically in the range of 1-10 pm; hence their immunodetection is challenging. ADAs toward Infliximab (IFX), a drug used to treat rheumatoid arthritis and other auto-immune diseases, are focussed. An ambipolar electrolyte-gated transistor (EGT) immunosensor is reported based on a reduced graphene oxide (rGO) channel and IFX bound to the gate electrode as the specific probe. The rGO-EGTs are easy to fabricate and exhibit low voltage operations (& LE; 0.3 V), a robust response within 15 min, and ultra-high sensitivity (10 am limit of detection). A multiparametric analysis of the whole rGO-EGT transfer curves based on the type-I generalized extreme value distribution is proposed. It is demonstrated that it allows to selectively quantify ADAs also in the co-presence of its antagonist tumor necrosis factor alpha (TNF-alpha), the natural circulating target of IFX.
2022
- Assessing the functional and structural stability of the Met80Ala mutant of cytochrome c in dimethylsulfoxide
[Articolo su rivista]
DI ROCCO, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio
abstract
The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and bi-osensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cyto-chrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscop-ically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the mod-ification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physi-ological milieu of cytochrome c.
2022
- Correction: Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF (Proceedings of the National Academy of Sciences of the United States of America (2019) 116 (3425-3430) DOI: 10.1073/pnas.1818003116)
[Articolo su rivista]
Jiang, X.; Burger, B.; Gajdos, F.; Bortolotti, C.; Futera, Z.; Breuer, M.; Blumberger, J.
abstract
Correction for "Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF," by Xiuyun Jiang, Bastian Burger, Fruzsina Gajdos, C. Bortolotti, Zdenek Futera, Marian Breuer, and Jochen Blumberger, which was first published February 12, 2019; 10.1073/pnas.1818003116 (Proc. Natl. Acad. Sci. U.S.A. 116, 3425-3430).
2022
- Detection of Neurofilament Light Chain with Label-Free Electrolyte-Gated Organic Field-Effect Transistors
[Articolo su rivista]
Solodka, K.; Berto, M.; Ferraro, D.; Menozzi, C.; Borsari, M.; Bortolotti, C. A.; Biscarini, F.; Pinti, M.
abstract
Neurofilaments are structural scaffolding proteins of the neuronal cytoskeleton. Upon axonal injury, the neurofilament light chain (NF-L) is released into the interstitial fluid and eventually reaches the cerebrospinal fluid and blood. Therefore, NF-L is emerging as a biomarker of neurological disorders, including neurodegenerative dementia, Parkinson's disease, and multiple sclerosis. It is challenging to quantify NF-L in bodily fluids due to its low levels. This work reports the detection of NF-L in aqueous solutions with an organic electronic device. The biosensor is based on the electrolyte-gated organic field-effect transistor (EGOFET) architecture and can quantify NF-L down to sub-pM levels; thanks to modification of the device gate with anti-NF-L antibodies imparted with potentially controlled orientation. The response is fitted to the Guggenheim–Anderson–De Boer adsorption model to describe NF-L adsorption at the gate/electrolyte interface, to consider the formation of a strongly adsorbed protein layer bound to the antibody and the formation of weakly bound NF-L multilayers, an interpretation which is also backed up by morphological characterization via atomic force microscopy. The label-free, selective, and rapid response makes this EGOFET biosensor a promising tool for the diagnosis and monitoring of neuronal damages through the detection of NF-L in physio-pathological ranges.
2022
- Laser Assisted Bioprinting of laminin on biodegradable PLGA substrates: Effect on neural stem cell adhesion and differentiation
[Articolo su rivista]
Tortorella, S.; Greco, P.; Valle, F.; Barbalinardo, M.; Foschi, G.; Lugli, F.; Dallavalle, M.; Zerbetto, F.; Bortolotti, C. A.; Biscarini, F.
abstract
Laser Assisted Bioprinting (LAB) is recognized to be an enabling and versatile microfabrication technology for regenerative medicine and artificial tissue engineering. Current bioprinting concentrates on a layer-by-layer approach to print cells in consecutive stacks or nets, to recreate specialized tissue functions with a top-down approach. This synthering of proximal cells however reduces the long range correlation of tissue parenchyma and stroma given by natural development, as result of cells mobility and signaling. In this work, laminin, one of the main components of brain extracellular matrix is deposited by LAB on a biodegradable scaffold made of poly(lactic-co-glycolic acid) (PLGA), providing chemical cues for the adhesion and differentiation of neural stem cells NE-4C induced by retinoic acid. Surface roughness and LAB induced aggregates promote the initial adhesion of neuronal stem cells to the PLGA substrate and influence the formation of clusters and interconnection between them. The amount of laminin delivered inside the spot area may be controlled down to sub-monolayer coverage and a positive correlation between the laminin spots and soma of trafficking cells is demonstrated, also by computational modelling. Anisotropic orientation of neurite outgrowth is induced upon differentiation, up to 70% of processes protruding from stem cell clusters. The comparative analysis shows that the topological cue plays a major role in enhabling cluster formation on the scaffold, but the bioprinted laminin spots appear to be regulating the strength of connection between them, opening the way to control the functional morphology of artificial neural tissue constructs.
2022
- Mixed ionic-electronic conductance across naphthalenediimide-functionalized biopolymers
[Articolo su rivista]
Ramanthrikkovil Variyam, A.; Agam, Y.; Paradisi, A.; Bortolotti, C. A.; Amdursky, N.
abstract
Mixed ionic-electronic conductive polymers are gaining high momentum for several electronic and bioelectronic applications. These polymers are composed of a synthetic conjugated polymer for electronic conduction and a synthetic ionomer for the ionic one. To make these materials more environmentally sustainable, much effort is being put in replacing synthetic polymers with biopolymers. However, to date, the only strategy for making mixed conductors with biopolymers is to blend them with synthetic polymers. Here, we show that by targeting certain amino acids of a protein-based biopolymer, we can modify them with naphthalenediimide (NDI) after polymerization, resulting in an improved electronic transport, which is in addition to the native ionic transport of the biopolymer. We further show that by reducing the NDI moieties we can reach conductivity values in the order of 40 mS cm−1, though NDI can re-oxidize depending on the environment of the biopolymer. The abundant nature of the protein building blocks together with the easy post-polymerization functionalization chemistry of NDI, which is very much different than any previous use of NDI in conductive polymers, is making our new strategy for making mixed ionic-electronic conductive biopolymers highly attractive.
2022
- Monitoring DNA Hybridization with Organic Electrochemical Transistors Functionalized with Polydopamine
[Articolo su rivista]
Sensi, M.; Migatti, G.; Beni, V.; D'Alvise, T. M.; Weil, T.; Berto, M.; Greco, P.; Imbriano, C.; Biscarini, F.; Bortolotti, C. A.
abstract
Organic electrochemical transistors (OECTs) are finding widespread application in biosensing, thanks to their high sensitivity, broad dynamic range, and low limit of detection. An OECT biosensor requires the immobilization of a biorecognition probe on the gate, or else on the channel, through several, often lengthy, chemical steps. In this work, a fast and straightforward way to functionalize the carbon gate of a fully screen-printed OECT by means of a polydopamine (PDA) film is presented. By chemical immobilization of an amine-terminated single-stranded oligonucleotide, containing the HSP70 promoter CCAAT sequence, on the PDA film, the detection of the complementary DNA strand is demonstrated. Furthermore, the specificity of the developed genosensor is assessed by comparing its response to the fully complementary strand with the one to partially complementary and noncomplementary oligonucleotides. The developed sensor shows a theoretical limit of detection (LOD) of 100 × 10−15 m and a dynamic range over four orders of magnitude.
2022
- Thermodynamics and Kinetics of Electron Transfer of 2 Electrode-Immobilized Small Laccase from Streptomyces coelicolor
[Articolo su rivista]
DI ROCCO, Giulia; Battistuzzi, Gianantonio; Ranieri, Antonio; Bortolotti, Carlo Augusto; Borsari, Marco; Sola, Marco
abstract
The thermodynamic and kinetic properties for the heterogeneous electron transfer (ET) were measured for the electrode-immobilized small laccase (SLAC) from Streptomyces coelicolor subjected to different electrostatic and covalent protein-electrode linkages, using cyclic voltammetry. Once immobilized electrostatically onto a gold electrode using mixed carboxyl- and hydroxy-terminated alkane-thiolate SAMs or covalently exploiting the same SAM subjected to N-hydroxysuccin-imide+1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS-EDC) chemistry, the SLAC-electrode electron flow occurs through the T1 center. The E°’ values (from +0.2 to +0.1 V vs. SHE at pH 7.0) are lower by more than 0.2 V compared to the protein either in solution or immobilized with different anchoring strategies using uncharged SAMs. For the present electrostatic and covalent binding, this effect can respectively be ascribed to the negative charge of the SAM surfaces and to deletion of the positive charge of Lys/Arg residues due to amide bond formation which both selectively stabilize the more positively charged oxidized SLAC. Observation of enthalpy/entropy compensation within the series indicates that the immobilized proteins experience different reduction-induced solvent reorganization effects. The E°’ values for the covalently attached SLAC are sensitive to three acid base equilibria, with apparent pKa values of pKa1ox =5.1, pKa1red=7.5, pKa2ox=8.4, pKa2red=10.9, pKa2ox=8.9, pKa2red=11.3 possibly involving one residue close to the T1 center and two residues (Lys and/or Arg) along with moderate protein unfolding, respectively. Therefore, the E°’ value of immobilized SLAC turns out to be particularly sensitive to the anchoring mode and me-30 dium conditions.
2021
- Activity and substrate specificity of lytic polysaccharide monooxygenases: An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida
[Articolo su rivista]
Serra, Ilenia; Piccinini, Daniele; Paradisi, Alessandro; Ciano, Luisa; Bellei, Marzia; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Sola, Marco; Walton, Paul H.; DI ROCCO, Giulia
abstract
Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward β-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic
resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.
2021
- Anti-drug antibody detection with label-free electrolyte-gated organic field-effect transistors
[Articolo su rivista]
Sensi, Matteo; Berto, Marcello; Gentile, Sara; Pinti, Marcello; Conti, Andrea; Pellacani, Giovanni; Salvarani, Carlo; Cossarizza, Andrea; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract
The efficacy of immunotherapy can be undermined by the development of an immune response against a drug/antibody mediated by anti-drug antibodies (ADAs) in treated patients. We present the first label-free EGOFET immunosensor that integrates a biological drug, Nivolumab (Opdivo©), as a specific recognition moiety to quantitatively and selectively detect ADAs against the drug. The limit of detection is 100 fM. This demonstration is a prelude to the detection of ADAs in a clinical setting in the treatment of different pathologies, and it also enables rapid screening of biological drugs for immunogenicity.
2021
- Electrolyte-gated transistors for enhanced performance bioelectronics
[Articolo su rivista]
Torricelli, Fabrizio; Adrahtas, Demetra Z.; Bao, Zhenan; Berggren, Magnus; Biscarini, Fabio; Bonfiglio, Annalisa; Bortolotti, Carlo A.; Frisbie, C. Daniel; Macchia, Eleonora; Malliaras, George G.; Mcculloch, Iain; Moser, Maximilian; Nguyen, Thuc-Quyen; Owens, Róisín M.; Salleo, Alberto; Spanu, Andrea; Torsi, Luisa
abstract
Electrolyte-gated transistors (EGTs), capable of transducing biological and biochemical inputs into amplified electronic signals and stably operating in aqueous environments, have emerged as fundamental building blocks in bioelectronics. In this Primer, the different EGT architectures are described with the fundamental mechanisms underpinning their functional operation, providing insight into key experiments including necessary data analysis and validation. Several organic and inorganic materials used in the EGT structures and the different fabrication approaches for an optimal experimental design are presented and compared. The functional bio-layers and/or biosystems integrated into or interfaced to EGTs, including self-organization and self-assembly strategies, are reviewed. Relevant and promising applications are discussed, including two-dimensional and three-dimensional cell monitoring, ultra-sensitive biosensors, electrophysiology, synaptic and neuromorphic bio-interfaces, prosthetics and robotics. Advantages, limitations and possible optimizations are also surveyed. Finally, current issues and future directions for further developments and applications are discussed.
2021
- Electron Transfer and Electrocatalytic Properties of the Immobilized Met80Ala Cytochrome c Variant in DMSO
[Articolo su rivista]
DI ROCCO, Giulia; Bighi, Beatrice; Borsari, Marco; Bortolotti, Carlo Augusto; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract
The electrode-immobilized Met80Ala variant of yeast iso-1 cytochrome c in mixed water/dimethylsulfoxide (DMSO) solutions up to 60 % v/v DMSO shows thermodynamic and kinetic parameters of electron exchange and electrocatalytic properties towards O2 reduction fully comparable to those in water. This is the result of moderate protein conformational changes thanks to immobilization that, to a certain extent, preserves protein structure, possibly due to the constraints on protein mobility/flexibility induced by the electrostatic interactions with the electrode-coating SAM. Upon increasing the DMSO content of the mixed solution beyond 60 %, a much larger perturbation occurs that leads to the progressive loss of the electrocatalytic ability. Therefore, under these conditions, the organic solvent remarkably affects the structure and properties of the protein probably involving major conformational changes or even the replacement of the 6th axial hydroxide
ligand of the heme iron with a strong protein ligand, possibly a lysine residue.
2021
- Green Fabrication of (6,5)Carbon Nanotube/Protein Transistor Endowed with Specific Recognition
[Articolo su rivista]
Berto, M.; Di Giosia, M.; Giordani, M.; Sensi, M.; Valle, F.; Alessandrini, A.; Menozzi, C.; Cantelli, A.; Gazzadi, G. C.; Zerbetto, F.; Calvaresi, M.; Biscarini, F.; Bortolotti, C. A.
abstract
A general single-step approach is introduced for the green fabrication of hybrid biosensors from water dispersion. The resulting device integrates the semiconducting properties of a carbon nanotube (CNT) and the functionality of a protein. In the initial aqueous phase, the protein (viz., lysozyme [LZ]) disperses the (6,5)CNT. Drop-casting of the dispersion on a test pattern (a silicon wafer with interdigitated Au source and drain electrodes) yields a fully operating, robust, electrolyte-gated transistor (EGT) in one step. The EGT response to biorecognition is then assessed using the LZ inhibitor N-acetyl glucosamine trisaccharide. Analysis of the output signal allows one to extract a protein-substrate binding constant in line with values reported for the free (without CNT) system. The methodology is robust, easy to optimize, redirectable toward different targets and sets the grounds for a new class of CNT-protein biosensors that overcome many limitations of the technology of fabrication of CNT biosensors.
2021
- How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing
[Articolo su rivista]
Ranieri, Antonio; Borsari, Marco; Casalini, Stefano; Di Rocco, Giulia; Sola, Marco; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio
abstract
Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.
2021
- Physical insights from the Frumkin isotherm applied to electrolyte gated organic transistors as protein biosensors
[Articolo su rivista]
Manco Urbina, Pamela Allison; Berto, Marcello; Greco, Pierpaolo; Sensi, Matteo; Borghi, Simone; Borsari, Marco; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract
2021
- Pseudoperoxidase activity, conformational stability and aggregation propensity of the His98Tyr myoglobin variant. Implications for the onset of myoglobinopathy.
[Articolo su rivista]
Hofbauer, Stefan; Pignataro, Marcello; Borsari, Marco; Bortolotti, Carlo; Di Rocco, Giulia; Ravenscroft, Gianina; Furtmüller, Paul; Obinger, Christian; Sola, Marco; Battistuzzi, Gianantonio
abstract
The autosomal dominant striated muscle disease myoglobinopathy is due to the single point mutation His98Tyr in human myoglobin (MB) [Olivè et al. Nat. Comm, 2019, 10, 1396], the heme-protein responsible for binding, storage and controlled release of O2 in striated muscle. In order to understand the molecular bases of this disease, a comprehensive biochemical and biophysical study on wt MB and the variant H98Y has been performed. Although only small differences exist between the active site architectures of the two proteins, the mutant exhibits an (i) increased reactivity towards hydrogen peroxide, (ii) a higher tendency to form high-molecular weight aggregates and (iii) is more prone to heme bleaching, possibly as a consequence of the observed H2O2-induced formation of the Tyr98 radical close to the metal center. These effects add to the impaired oxygen binding capacity and faster heme dissociation of the H98Y variant compared to wt MB. As the above effects result from bond formation/cleavage events occurring at the distal and proximal heme sites, it appears that the molecular determinants of the disease are localized there. These findings set the bases for clarifying the onset of the cascade of chemical events that are responsible for the pathological symptoms of myoglobinopathy.
2021
- Sensing Inflammation Biomarkers with Electrolyte-Gated Organic Electronic Transistors
[Articolo su rivista]
Burtscher, B.; Manco Urbina, P. A.; Diacci, C.; Borghi, S.; Pinti, M.; Cossarizza, A.; Salvarani, C.; Berggren, M.; Biscarini, F.; Simon, D. T.; Bortolotti, C. A.
abstract
An overview of cytokine biosensing is provided, with a focus on the opportunities provided by organic electronic platforms for monitoring these inflammation biomarkers which manifest at ultralow concentration levels in physiopathological conditions. Specifically, two of the field's state-of-the-art technologies—organic electrochemical transistors (OECTs) and electrolyte gated organic field effect transistors (EGOFETs)—and their use in sensing cytokines and other proteins associated with inflammation are a particular focus. The overview will include an introduction to current clinical and “gold standard” quantification techniques and their limitations in terms of cost, time, and required infrastructure. A critical review of recent progress with OECT- and EGOFET-based protein biosensors is presented, alongside a discussion onthe future of these technologies in the years and decades ahead. This is especially timely as the world grapples with limited healthcare diagnostics during the Coronavirus disease (COVID-19)pandemic where one of the worst-case scenarios for patients is the “cytokine storm.” Clearly, low-cost point-of-care technologies provided by OECTs and EGOFETs can ease the global burden on healthcare systems and support professionals by providing unprecedented wealth of data that can help to monitor disease progression in real time.
2021
- The Enthalpic and Entropic Terms of the Reduction Potential of Metalloproteins: Determinants and Interplay
[Articolo su rivista]
Di Rocco, G.; Battistuzzi, G.; Borsari, M.; Bortolotti, C. A.; Ranieri, A.; Sola, M.
abstract
Splitting the reduction potential of electron transport (ET) proteins and redox metalloenzymes into the enthalpic and entropic contributions is an insightful practice to gaining insight into the molecular determinants of the thermodynamic propensity of the metal center to accept or release electrons. The strict control of such propensity is essential for the functioning of the electron transport chains in bioenergetics and the organized network of the countless reactions of the redox metabolism in all organisms. Here, the first comprehensive overview is offered on the thermodynamic data obtained in the last three decades for the main classes of ET species, namely c-type cytochromes and proteins containing T1 copper and iron-sulfur centers, along with some heme metalloenzymes. These families show many common features in the balance of the enthalpic and entropic terms, which will be brought to light. The enthalpic terms related to ligation features in the first coordination sphere of the metal and weak binding and electrostatics in the surrounding matrix do count a lot in this balance. Reduction entropy is much less important that it would appear from the raw thermodynamic data, particularly for electron transport (ET) metalloproteins. This is due to reduction-induced solvent-related molecular events which dominate the measured entropy changes but affect much less the reduction free energy due to the compensatory effects of the associated enthalpic terms (a phenomenology known as enthalpy-entropy compensation, EEC). Thus the entropy changes seldom exert a real influence on the E°’ of metalloredox proteins; this is restricted to metal sites subjected to reduction-induced protein-based changes in the accessible configurational microstates. It follows that in most cases, especially for ET species, the E° changes due to point mutations, ligand binding and charge changes have and ultimate enthalpic origin. Hence, they should be accounted for with coordination chemistry and electrostatics notions. Only if they don’t, protein-based entropic effects could play a role. In this review, we go through the data gathered for the main classes of ET species and heme enzymes that brought us to this conclusion.
2020
- A Bacterial Photosynthetic Enzymatic Unit Modulating Organic Transistors with Light
[Articolo su rivista]
Di Lauro, M.; la Gatta, S.; Bortolotti, C. A.; Beni, V.; Parkula, V.; Drakopoulou, S.; Giordani, M.; Berto, M.; Milano, F.; Cramer, T.; Murgia, M.; Agostiano, A.; Farinola, G. M.; Trotta, M.; Biscarini, F.
abstract
The photochemical core of every photosynthetic apparatus is the reaction center, a transmembrane enzyme that converts photons into charge-separated states across the biological membrane with an almost unitary quantum yield. A light-responsive organic transistor architecture, which converts light into electrical current by exploiting the efficiency of this biological machinery, is presented. Proper surface tailoring enables the integration of the bacterial reaction center as photoactive element in organic transistors, allowing the transduction of its photogenerated voltage into photomodulation of the output current up to two orders of magnitude. This device architecture, termed light-responsive electrolyte-gated organic transistor, is the prototype of a new generation of low-power hybrid bio-optoelectronic organic devices.
2020
- Adsorbing surface strongly influences the pseudoperoxidase and nitrite reductase activity of electrode-bound yeast cytochrome c. The effect of hydrophobic immobilization.
[Articolo su rivista]
Lancellotti, Lidia; Borsari, Marco; Bonifacio, Alois; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Casalini, Stefano; Ranieri, Antonio; Battistuzzi, Gianantonio; Sola, Marco
abstract
The Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) and their adducts with cardiolipin immobilized onto a gold electrode coated with a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol were studied through cyclic voltammetry and surface-enhanced resonance Raman spectroscopy (SERRS). The electroactive species - containing a six-coordinate His/His axially ligated heme and a five-coordinate His/- heme stable in the oxidized and reduced state, respectively - and the pseudoperoxidase activity match those found previously for the wt species and are only slightly affected by CL binding. Most importantly, the reduced His/- ligated form of these variants is able to catalytically reduce the nitrite ion, while electrode-immobilized wt ycc and other His/Met heme ligated variants under a variety of conditions are not. Besides the pseudoperoxidase and nitrite reductase functions, which are the most physiologically relevant abilities of these constructs, also axial heme ligation and the equilibria between conformers are strongly affected by the nature - hydrophobic vs. electrostatic - of the non-covalent interactions determining protein immobilization. Also affected are the catalytic activity changes induced by a given mutation as well as those due to partial unfolding due to CL binding. It follows that under the same solution conditions the structural and functional properties of immobilized ycc are surface-specific and therefore cannot be transferred from an immobilized system to another involving different interfacial protein-SAM interactions.
2020
- Binding of S. cerevisiae iso‑1 cytochrome c and its surface lysine‑to‑alanine variants to cardiolipin: charge effects and the role of the lipid to protein ratio
[Articolo su rivista]
Paradisi, Alessandro; Bellei, Marzia; Paltrinieri, Licia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Battistuzzi, Gianantonio
abstract
The interaction of cytochrome c with cardiolipin (CL) is a critical step in the initial stages of apoptosis and is mediated by
a positively charged region on the protein surface comprising several lysine residues (site A). Here, the interaction of wt S.
cerevisiae cytochrome c (ycc) and its K72A/K73A, K72A/K79A, K73A/K79A and K72A/K73A/K79A variants with CL
was studied through UV–Vis and MCD spectroscopies at pH 7 and molecular dynamics (MD) simulations, to clarify the role
of the mutated lysines. Moreover, the influence of the lipid to protein ratio on the interaction mechanism was investigated
using low (0.5–10) and high (5–60) CL/ycc molar ratios, obtained with small and gradual or large and abrupt CL additions,
respectively. Although all proteins bind to CL, switching from the native low-spin His/Met-ligated form to a low-spin bis-His
conformer and to a high-spin species at larger CL concentrations, the two schemes of CL addition show relevant differences
in the CL/ycc molar ratios at which the various conformers appear, due to differences in the interaction mechanism. Extended
lipid anchorage and peripheral binding appear to prevail at low and high CL/ycc molar ratios, respectively. Simultaneous
deletion of two or three surface positive charges from Site A does not abolish CL binding, but instead increases protein affinity
for CL. MD calculations suggest this unexpected behavior results from the mutation-induced severe weakening of the
H-bond connecting the Nε
of His26 with the backbone oxygen of Glu44, which lowers the conformational stability compared
to the wt species, overcoming the decreased surface electrostatic interaction.
2020
- Electrochemical data on redox properties of human Cofilin-2 and its Mutant S3D
[Articolo su rivista]
Pignataro, M.; Di Rocco, G.; Lancellotti, L.; Bernini, F.; Subramanian, K.; Castellini, E.; Bortolotti, C. A.; Malferrari, D.; Moro, D.; Valdrè, G.; Borsari, M.; del Monte, F.
abstract
The reported data are related to a research paper entitled "Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation" [1]. Info about the formation and redox properties of the disulfide bridge of a protein is quite difficult to obtain and only in a few cases was it possible to observe a cyclic voltammetry (CV) signal [2,3]. Human cofilin-2 contains two cysteines (Cys39 and Cys80) which can be oxidized in suitable conditions and form a disulfide bridge [1]. For this purpose, CV measurements were carried out on human cofilin-2 WT and its mutant S3D immobilized on a gold electrode coated by an anionic self-assembled monolayer (SAM), after a pre-oxidation time which was fundamental for observing a CV signal relating to the oxidation/reduction process of the disulfide bridge of the proteins. The data include CV curves obtained with and without electrochemical pre-oxidation and after oxidation with H2O2. In addition, the plot of the cathodic peak current vs. electrochemical pre-oxidation time and the pH dependence of the formal potential (E°’) are reported. The data obtained by CV measurements were used to determine the time required to form the disulfide bridge for the immobilized proteins and, consequently, to observe the CV signal, to calculate the E°’ values and analyse the pH dependence of E°’. The electrochemical data were provided which will be useful for further electrochemical investigations regarding proteins bearing disulfide bridge(s) or cysteines prone to oxidation.
2020
- Flexible Printed Organic Electrochemical Transistors for the Detection of Uric Acid in Artificial Wound Exudate
[Articolo su rivista]
Galliani, M.; Diacci, C.; Berto, M.; Sensi, M.; Beni, V.; Berggren, M.; Borsari, M.; Simon, D. T.; Biscarini, F.; Bortolotti, C. A.
abstract
Low-cost, minimally invasive sensors able to provide real-time monitoring of wound infection can enable the optimization of healthcare resources in chronic wounds management. Here, a novel printed organic electrochemical transistors (OECT) biosensor for monitoring uric acid (UA), a bacterial infection biomarker in wounds, is demonstrated in artificial wound exudate. The sensor exploits the enzymatic conversion of UA to 5-hydroxyisourate, catalyzed by Uricase entrapped in a dual-ionic-layer hydrogel membrane casted onto the gate. The sensor response is based on the catalytic oxidation of the hydrogen peroxide, generated as part of the Uricase regeneration process, at the Pt modified gate. The proposed dual membrane avoids the occurrence of nonspecific faradic reactions as, for example, the direct oxidation of UA or other electroactive molecules that would introduce a potentially false negative response. The biosensor is robust and its response is reproducible both in phosphate buffer saline and in complex solutions mimicking the wound exudate. The sensor has a high sensitivity in the range encompassing the pathological levels of UA in wounds (<200 μm) exhibiting a limit of detection of 4.5 μm in artificial wound exudate. All these characteristics make this OECT-based biosensor attractive for wound monitoring interfaced to the patient.
2020
- Harnessing Selectivity and Sensitivity in Electronic Biosensing: A Novel Lab-on-Chip Multigate Organic Transistor
[Articolo su rivista]
Parkula, Vitaliy; Berto, Marcello; Diacci, Chiara; Patrahau, Bianca; Di Lauro, Michele; Kovtun, Alessandro; Liscio, Andrea; Sensi, Matteo; Samorì, Paolo; Greco, Pierpaolo; Bortolotti, Carlo A; Biscarini, Fabio
abstract
Electrolyte gated organic transistors can operate as powerful ultrasensitive biosensors, and efforts are currently devoted to devising strategies for reducing the contribution of hardly avoidable, nonspecific interactions to their response, to ultimately harness selectivity in the detection process. We report a novel lab-on-a-chip device integrating a multigate electrolyte gated organic field-effect transistor (EGOFET) with a 6.5 μL microfluidics set up capable to provide an assessment of both the response reproducibility, by enabling measurement in triplicate, and of the device selectivity through the presence of an internal reference electrode. As proof-of-concept, we demonstrate the efficient operation of our pentacene based EGOFET sensing platform through the quantification of tumor necrosis factor alpha with a detection limit as low as 3 pM. Sensing of inflammatory cytokines, which also include TNFα, is of the outmost importance for monitoring a large number of diseases. The multiplexable organic electronic lab-on-chip provides a statistically solid, reliable, and selective response on microliters sample volumes on the minutes time scale, thus matching the relevant key-performance indicators required in point-of-care diagnostics.
2020
- Met80 and Tyr67 affect the chemical unfolding of yeast cytochrome c: comparing solution vs. immobilized state
[Articolo su rivista]
Paradisi, Alessandro; Lancellotti, Lidia; Borsari, Marco; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract
Urea-induced denaturation of the Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) was studied through variable temperature diffusive cyclic voltammetry and electronic absorption, CD and MCD spectroscopies. The susceptibility to unfolding of both variants - represented by the free energy of unfolding at denaturant infinite dilution, ∆〖G°〗_u^(H_2 O)is greater compared to the species showing an intact Met/His coordination, as observed previously for the same species immobilized onto a functionalized electrode. This is consistent with the role of the axial Fe-(S)Met bond and the H-bond network involving Tyr67 in stabilizing the polypeptide matrix in the heme crevice. Notably, we find that the unfolding propensity and axial heme iron coordination of the present Fe-(S)Met bond-deprived variants is affected by the motional regime of the protein. In particular, electrostatic adsorption onto a negatively charged SAM surface - that would mimic the phospholipidic inner mitochondrial membrane - facilitates unfolding compared to the solution state, especially at room temperature. This finding has a physiological relevance related to the cytochrome c interaction with cardiolipin at the IMM in the early stages of apoptosis. Moreover, while both immobilized variants maintain the His/OH- axial heme iron coordination up to 7 M urea, the same species in solution are subjected to urea-induced replacement of the axial hydroxide ligand by a His ligand. The contribution of the enthalpic and entropic terms to ∆〖G°〗_u^(H_2 O) were found to be opposite (H-S compensation) indicating that unfolding thermodynamics are strongly affected by changes in the hydrogen bonding network in the hydration sphere of the protein.
2020
- Neuromorphic Organic Devices: Neuromorphic Organic Devices that Specifically Discriminate Dopamine from Its Metabolites by Nonspecific Interactions (Adv. Funct. Mater. 28/2020)
[Altro]
Giordani, Martina; Sensi, Matteo; Berto, Marcello; Di Lauro, Michele; Bortolotti, Carlo Augusto; Gomes, Henrique Leonel; Zoli, Michele; Zerbetto, Francesco; Fadiga, Luciano; Biscarini, Fabio
abstract
2020
- Neuromorphic Organic Devices that Specifically Discriminate Dopamine from Its Metabolites by Nonspecific Interactions
[Articolo su rivista]
Giordani, M.; Sensi, M.; Berto, M.; Di Lauro, M.; Bortolotti, C. A.; Gomes, H. L.; Zoli, M.; Zerbetto, F.; Fadiga, L.; Biscarini, F.
abstract
Specific detection of dopamine (DA) is achieved with organic neuromorphic devices with no specific recognition function in an electrolyte solution. The response to voltage pulses consists of amplitude-depressed current spiking mimicking the short-term plasticity (STP) of synapses. An equivalent circuit hints that the STP timescale of the device arises from the capacitance and resistance of the poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) in series with the electrolyte resistance. Both the capacitance and resistance of PEDOT:PSS change with solution compositions. Dose curves are constructed from the STP timescale for each DA metabolite from pM to mM range of concentrations. The STP response of DA is distinctive from the other metabolites even when differences are by one functional group. Both STP and sensitivity to DA are larger across the patho-physiological range with respect to those to DA metabolites. Density functional theory calculations hint to a stronger hydrogen bond pattern of DA ammonium compared to cationic metabolites. The exponential correlation between STP and the binding energy of DA metabolites interacting with PEDOT:PSS indicates that the slow dynamics of ionic species in and out PEDOT:PSS is the origin of the neuromorphic STP. The sensing framework discriminates differences of nonspecific interactions of few kcal mol−1, corresponding to one functional group in the molecule.
2020
- Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation
[Articolo su rivista]
Pignataro, M.; Di Rocco, G.; Lancellotti, L.; Bernini, F.; Subramanian, K.; Castellini, E.; Bortolotti, C. A.; Malferrari, D.; Moro, D.; Valdre, G.; Borsari, M.; Monte, F. D.
abstract
Cofilins are small protein of the actin depolymerizing family. Actin polymerization/depolymerization is central to a number of critical cellular physiological tasks making cofilin a key protein for several physiological functions of the cell. Cofilin activity is mainly regulated by phosphorylation on serine residue 3 making this post-translational modification key to the regulation of myofilament integrity. In fact, in this form, the protein segregates in myocardial aggregates in human idiopathic dilated cardiomyopathy. Since myofilament network is an early target of oxidative stress we investigated the molecular changes induced by oxidation on cofilin isoforms and their interplay with the protein phosphorylation state to get insight on whether/how those changes may predispose to early protein aggregation. Using different and complementary approaches we characterized the aggregation properties of cofilin-2 and its phosphomimetic variant (S3D) in response to oxidative stress in silico, in vitro and on isolated cardiomyocytes. We found that the phosphorylated (inactive) form of cofilin-2 is mechanistically linked to the formation of an extended network of fibrillar structures induced by oxidative stress via the formation of a disulfide bond between Cys39 and Cys80. Such phosphorylation-dependent effect is likely controlled by changes in the hydrogen bonding network involving Cys39. We found that the sulfide ion inhibits the formation of such structures. This might represent the mechanism for the protective effect of the therapeutic agent Na2S on ischemic injury.
2020
- The biology of Lonp1: More than a mitochondrial protease
[Capitolo/Saggio]
Gibellinia, Lara; De Gaetano, Anna; Mandrioli, Mauro; Van Tongeren, Elia; Bortolotti, Carlo Augusto; Cossarizza, Andrea; Pinti, Marcello
abstract
Initially discovered as a protease responsible for degradation of misfolded or damaged proteins, the mitochondrial Lon protease (Lonp1) turned out to be a multifaceted enzyme, that displays at least three different functions (proteolysis, chaperone activity, binding of mtDNA) and that finely regulates several cellular processes, within and without mitochondria. Indeed, LONP1 in humans is ubiquitously expressed, and is involved in regulation of response to oxidative stress and, heat shock, in the maintenance of mtDNA, in the regulation of mitophagy. Furthermore, its proteolytic activity can regulate several biochemical pathways occurring totally or partially within mitochondria, such as TCA cycle, oxidative phosphorylation, steroid and heme biosynthesis and glutamine production. Because of these multiple activities, Lon protease is highly conserved throughout evolution, and mutations occurring in its gene determines severe diseases in humans, including a rare syndrome characterized by Cerebral, Ocular, Dental, Auricular and Skeletal anomalies (CODAS). Finally, alterations of LONP1 regulation in humans can favor tumor progression and aggressiveness, further highlighting the crucial role of this enzyme in mitochondrial and cellular homeostasis.
2020
- Urea-induced denaturation of immobilized yeast iso-1 cytochrome c: role of Met80 and Tyr67 in the thermodynamics of unfolding and promotion of pseudoperoxidase and nitrite reductase activities
[Articolo su rivista]
Lancellotti, Lidia; Borsari, Marco; Bellei, Marzia; Bonifacio, Alois; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract
The Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc ) immobilized on a decane-1-thiol coated gold electrode subjected to the denaturing action of urea were studied through variable temperature cyclic voltammetry and Surface-Enhanced Resonance Raman spectroscopy (SERRS). We found that the His/OH - axial heme iron coordination in both variants is unaffected by urea up to 7 M, although some conformational changes occur that increase exposure of the heme center to solvent. The thermodynamics of the unfolding process were determined with an unprecedented approach, which can be of general use for electroactive proteins. The free energy of unfolding for both variants includes relevant entropic contributions and is lower than that for the species carrying an intact Met/His coordination, consistent with the role of the axial Fe-(S)Met bond and the H-bond network involving Tyr67 in stabilizing the polypeptide matrix in the heme crevice. Their
lower conformational stability results in a different interaction with the MUA/MU SAM compared to the His/Met ycc forms. Denaturation invariably slows down the heterogeneous electron transfer process, but its effect on the activation enthalpy and
pre-exponential factor differs for the species with and without His/Met axial heme ligation. In particular, urea unfolding of the M80A and M80A/Y67A mutants lowers the structural restraint to the heterogeneous ET. Here we show that removal of the Met
ligand and an increased accessibility of the heme center to solvent through partial protein unfolding– which mimic the molecular stress experienced by mammalian cytochromes c upon binding to cardiolipin in the early events of apoptosis - add up to transform cytochrome c into an efficient electrocatalyst toward the reduction of hydrogen peroxide and nitrite.
2019
- Biosensing with Electrolyte Gated Organic Field Effect Transistors
[Capitolo/Saggio]
Bortolotti, Carlo Augusto; Berto, Marcello; Sensi, Matteo; DI LAURO, Michele; Biscarini, Fabio
abstract
Electrolyte Gated Organic Field Effect Transistors (EGOFETs) are rapidly emerging as novel players in the field of biosensing: they allow ultra-sensitive, label-free and fast response, and can be employed to sense very diverse analytes, from small molecules to large multimeric proteins. Here, we present the current level of understanding of the working mechanism of EGOFETs, and review some of the most recent and relevant applications as sensors for healthcare and life sciences, discussing advantages and limitations of this technology. EGOFETs appear as a powerful sensing platform that can be readily adapted to the detection of a wide range of biologically relevant species.
2019
- EGOFET Gated by a Molecular Electronic Switch: A Single-Device Memory Cell
[Articolo su rivista]
Parkula, V.; Maglione, M. S.; Casalini, S.; Zhang, Q.; Greco, P.; Bortolotti, C. A.; Rovira, C.; Mas-Torrent, M.; Biscarini, F.
abstract
Electrolyte-gated organic field-effect transistors (EGOFETs) exploit the transduction of interfacial phenomena, such as biorecognition or redox processes, into detectable changes of electrical response. Here, it is shown that, beyond sensing applications, EGOFETs may act effectively as memory devices, through the functionalization of the gate electrode with a self-assembly monolayer comprising a switching molecule undergoing a large and persistent change of dipole moment, upon application of a small (0.6 V) programming potential. This first example of a switchable EGOFET device with memory retention is based on a tetrathiafulvalene derivative self-assembled on gold and an aqueous buffer as electrolyte in a microfluidic assembly. Changes of the self-assembled monolayer redox state lead to variations of the gate electrochemical potential and, as a consequence, the EGOFET's threshold voltage undergoes reversible shifts larger than 100 mV. The distinctive electrical readout upon different redox states opens the possibility of writing and erasing information, thus making the transistor behave as a single memory cell.
2019
- Electrocatalytic Properties of Immobilized Heme Proteins: Basic Principles and Applications
[Articolo su rivista]
Ranieri, Antonio; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Battistuzzi, Gianantonio; Sola, Marco; Borsari, Marco
abstract
Heme proteins encompass redox enzymes, electron transferases, and species for dioxygen transport and storage. Upon immobilization on a conductive surface, heme proteins can accomplish bioelectrocatalysis. In this process, they carry out oxidation or reduction of substrates at a solid electrode acting as electron acceptor or donor, respectively, thanks to electron transfer processes occurring at the interphase. The efficiency of bioelectrocatalysis depends on the electrical communication of the protein with the electrode surface, retention of protein structure upon adsorption and accessibility of the substrate to the active site. This Minireview outlines the main factors affecting bioelectrocatalysis by adsorbed heme proteins, highlights open issues, and summarizes recent advances in the field.
2019
- Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF
[Articolo su rivista]
Jiang, X.; Burger, B.; Gajdos, F.; Bortolotti, C.; Futera, Z.; Breuer, M.; Blumberger, J.
abstract
The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an ≈ 10 3 -fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.
2019
- Label free detection of plant viruses with organic transistor biosensors
[Articolo su rivista]
Berto, Marcello; Vecchi, Eugenia; Baiamonte, Luca; Condò, Carla; Sensi, Matteo; Di Lauro, Michele; Sola, Marco; De Stradis, Angelo; Biscarini, Fabio; Minafra, Angelantonio; Bortolotti, Carlo Augusto
abstract
Plum Pox Virus (PPV) is the pathogen responsible for Sharka, a highly infectious disease affecting stone fruit trees and causing severe economic damages, which can be only contained through early-detection and frequent monitoring. We propose a bioelectronic PPV biosensor, based on a electrolyte-gated organic field-effect transistor (EGOFET), for the specific detection of PPV in plant extracts with a sub ng/ml detection limit. The sensing unit of the biosensor is based on anti-PPV antibodies, uniformly oriented on the gold gate electrode by using a sub-monolayer of Protein G. The sensitivity and dynamic range of the EGOFET-based biosensor are comparable to those of commercially available platforms for detection of plant pathogens. This novel electronic immunosensor is compatible with low-cost fabrication procedures and can be easily reconfigured into a fully portable device to be operated in greenhouse and in the field orchards.
2019
- Modulating the Faradic Operation of All-Printed Organic Electrochemical Transistors by Facile in Situ Modification of the Gate Electrode
[Articolo su rivista]
Sensi, Matteo; Berto, Marcello; Candini, Andrea; Liscio, Andrea; Cossarizza, Andrea; Beni, Valerio; Biscarini, Fabio; Bortolotti, Carlo Augusto
abstract
Organic electrochemical transistors (OECTs) operated in the faradic regime were shown as outperforming transducers of bioelectric signals in vitro and in vivo. Fabrication by additive manufacturing techniques fosters OECTs as ideal candidates for point-of-care applications, as well as imposes limitations on the choice of materials and their processing conditions. Here, we address the question of how the response of fully printed OECTs depends on gate electrode material. Toward this end, we investigate the redox processes underlying the operation of OECTs under faradic regime, to show OECTs with carbon gate (C-gate) that exhibit no current modulation gate voltages <1.2 V. This is a hallmark that no interference with the faradic operation of the device enabled by redox processes occurs when operating C-gate OECTs in the low-voltage range as label-free biosensors for the detection of electroactive (bio)molecules. To tune the faradic response of the device, we electrodeposited Au on the carbon gate (Au-C-gate), obtaining a device that operates at lower gate voltage values than C-gate OECT. The presence of gold on the gate allowed further modification of the electrical performances by functionalization of the Au-C-gate with different self-assembled monolayers by fast potential-pulse-assisted method. Moreover, we show that the presence in the electrolyte solution of an external redox probe can be used to drive the faradic response of both C- and Au-C-gate OECTs, impacting on the gate potential window that yields effective drain current modulation. The results presented here suggest possible new strategies for controlling the faradic operation regime of OECTs sensors by chemical modification of the gate surface.
2019
- Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions
[Articolo su rivista]
Olivé, Montse; Engvall, Martin; Ravenscroft, Gianina; Cabrera-Serrano, Macarena; Jiao, Hong; Bortolotti, Carlo Augusto; Pignataro, Marcello; Lambrughi, Matteo; Jiang, Haibo; Forrest, Alistair R. R.; Benseny-Cases, Núria; Hofbauer, Stefan; Obinger, Christian; Battistuzzi, Gianantonio; Bellei, Marzia; Borsari, Marco; Di Rocco, Giulia; Viola, Helena M.; Hool, Livia C.; Cladera, Josep; Lagerstedt-Robinson, Kristina; Xiang, Fengqing; Wredenberg, Anna; Miralles, Francesc; José Baiges, Juan; Malfatti, Edoardo; Romero, Norma B.; Streichenberger, Nathalie; Vial, Christophe; Claeys, Kristl G.; Straathof, Chiara S. M.; Goris, An; Freyer, Christoph; Lammens, Martin; Bassez, Guillaume; Kere, Juha; Clemente, Paula; Sejersen, Thomas; Udd, Bjarne; Vidal, Noemí; Ferrer, Isidre; Edström, Lars; Wedell, Anna; Laing, Nigel G.
abstract
Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T (p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.
2018
- Bi-allelic mutations in MYL1 cause a severe congenital myopathy
[Articolo su rivista]
Ravenscroft, G.; Zaharieva, I. T.; Bortolotti, C. A.; Lambrughi, M.; Pignataro, M.; Borsari, M.; Sewry, C. A.; Phadke, R.; Haliloglu, G.; Ong, R.; Goullee, H.; Whyte, T.; Consortium, U. K.; Manzur, A.; Talim, B.; Kaya, U.; Osborn, D. P. S.; Forrest, A. R. R.; Laing, N. G.; Muntoni, F.
abstract
Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, ∼50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. We performed exome sequencing on two consanguineous probands diagnosed with a congenital myopathy and muscle biopsy showing selective atrophy/hypotrophy or absence of type II myofibres. We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain (ELC) in both probands. A homozygous essential splice acceptor variant (c.479-2A > G, predicted to result in skipping of exon 5 was identified in Proband 1, and a homozygous missense substitution (c.488T>G, p.(Met163Arg)) was identified in Proband 2. Protein modelling of the p.(Met163Arg) substitution predicted it might impede intermolecular interactions that facilitate binding to the IQ domain of myosin heavy chain, thus likely impacting on the structure and functioning of the myosin motor. MYL1 was markedly reduced in skeletal muscle from both probands, suggesting that the missense substitution likely results in an unstable protein. Knock down of myl1 in zebrafish resulted in abnormal morphology, disrupted muscle structure and impaired touch-evoked escape responses, thus confirming that skeletal muscle fast-twitch specific myosin ELC is critical for myofibre development and function. Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with severe congenital myopathy.
2018
- Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations
[Articolo su rivista]
Bertini, Luca; Breglia, Raffaella; Lambrughi, Matteo; Fantucci, Piercarlo; De Gioia, Luca; Borsari, Marco; Sola, Marco; Bortolotti, Carlo Augusto; Bruschi, Maurizio
abstract
Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.
2018
- Core-rod myopathy due to a novel mutation in BTB/POZ domain of KBTBD13 manifesting as late onset LGMD
[Articolo su rivista]
Garibaldi, Matteo; Fattori, Fabiana; Bortolotti, Carlo Augusto; Brochier, Guy; Labasse, Clemence; Verardo, Margherita; Servian-Morilla, Emilia; Gibellini, Lara; Pinti, Marcello; Di Rocco, Giulia; Raffa, Salvatore; Pennisi Elena, Maria; Bertini Enrico, Silvio; Paradas, Carmen; Romero Norma, Beatriz; Antonini, Giovanni.
abstract
Few genes (RYR1, NEB, ACTA1, CFL2, KBTBD13) have been associated with core-rod congenital myopathies [7]. KBTBD13 belongs to the Kelch-repeat super-family of proteins and is implicated in the ubiquitination pathway. Dominant mutations in KBTBD13 have been associated with a peculiar form of core-rod myopathy (NEM6) so far [10]. Childhood onset, slowly progressive proximal muscle weakness with characteristic slowness of movements and combination of nemaline rods, irregular shaped cores and unusual type2 fibres hypotrophy at muscle biopsy, were the main characteristics shared in all the affected members of the four KBTBD13 families reported in the literature [12].
We report on a 65 years old patient, of Sardinian origin, with atypical clinical and morphological presentation of NEM6 due to a novel mutation in KBTBD13 gene.
2018
- EGOFET Peptide Aptasensor for Label-Free Detection of Inflammatory Cytokines in Complex Fluids
[Articolo su rivista]
Berto, Marcello; Diacci, Chiara; D'Agata, Roberta; Pinti, Marcello; Bianchini, Elena; Lauro, Michele Di; Casalini, Stefano; Cossarizza, Andrea; Berggren, Magnus; Simon, Daniel; Spoto, Giuseppe; Biscarini, Fabio; Bortolotti, Carlo A.
abstract
Organic electronic transistors are rapidly emerging as ultrahigh sensitive
label-free biosensors suited for point-of-care or in-field deployed applications.
Most organic biosensors reported to date are based on immunorecognition
between the relevant biomarkers and the immobilized antibodies, whose use
is hindered by large dimensions, poor control of sequence, and relative instability.
Here, an electrolyte-gated organic field effect transistor (EGOFET) biosensor
where the recognition units are surface immobilized peptide aptamers
(Affimer proteins) instead of antibodies is reported. Peptide aptasensor for
the detection of the pro-inflammatory cytokine tumor necrosis factor alpha
(TNFα) with a 1 × 10−12 M limit of detection is demonstrated. Ultralow sensitivity
is met even in complex solutions such as cell culture media containing
10% serum, demonstrating the remarkable ligand specificity of the device.
The device performances, together with the simple one-step immobilization
strategy of the recognition moieties and the low operational voltages, all
prompt EGOFET peptide aptasensors as candidates for early diagnostics and
monitoring at the point-of-care.
2018
- Exploiting interfacial phenomena in organic bioelectronics: Conformable devices for bidirectional communication with living systems
[Articolo su rivista]
Di Lauro, Michele; Benaglia, Simone; Berto, Marcello; Bortolotti, Carlo A.; Zoli, Michele; Biscarini, Fabio
abstract
A novel fully organic bioelectronic device is presented and validated as electronic transducer and current stimulator for brain implants. The device integrates polymeric electrodes made of poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) on paper thin foils, resulting in a high surface-to-volume ratio architecture that exhibits high sensitivity to interfacial ionic transport phenomena. The prototyping technique herein presented yields devices for the bidirectional communication with biological systems whose dimensionality can be controlled according to the desired application. Transduction of ultra-low local-field potentials and delivery of voltage pulse-trains alike those used in deep-brain stimulation are herein assessed, paving the way towards novel theranostic strategies for the treatment of Parkinson's Disease and other severe neurodegenerative and/or traumatic pathologies of the central nervous system.
2018
- Label free urea biosensor based on organic electrochemical transistors
[Articolo su rivista]
Berto, Marcello; Diacci, Chiara; Theuer, Lorenz; Di Lauro, Michele; Simon, Daniel T.; Berggren, Magnus; Biscarini, Fabio; Beni, Valerio; Bortolotti, Carlo A.
abstract
The quantification of urea is of the utmost importance not only in medical diagnosis, where it serves as a potential indicator of kidney and liver disfunction, but also in food safety and environmental control. Here, we describe a urea biosensor based on urease entrapped in a crosslinked gelatin hydrogel, deposited onto a fully printed PEDOT:PSS-based organic electrochemical transistor (OECT). The device response is based on the modulation of the channel conductivity by the ionic species produced upon urea hydrolysis catalyzed by the entrapped urease. The biosensor shows excellent reproducibility, a limit of detection as low as 1 μM and a response time of a few minutes. The fabrication of the OECTs by screen-printing on flexible substrates ensures a significant reduction in manufacturing time and costs. The low dimensionality and operational voltages (0.5 V or below) of these devices contribute to make these enzymatic OECT-based biosensors as appealing candidates for high-throughput monitoring of urea levels at the point-of-care or in the field.
2018
- The influence of the Cys46/Cys55 disulfide bond on the redox and spectroscopic properties of human neuroglobin.
[Articolo su rivista]
Bellei, Marzia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Borsari, Marco; Lancellotti, Lidia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract
Neuroglobin is a monomeric globin containing a six-coordinate heme b, expressed in the nervous system, which exerts an important neuroprotective role. In the human protein (hNgb), Cys46 and Cys55 form an intramolecular disulfide bond under oxidizing conditions, whose cleavage induces a helix-to-strand rearrangement of the CD loop that strengthens the bond between the heme iron and the distal histidine. Hence, it is conceivable that the intramolecular disulfide bridge modulates the functionality of human neuroglobin by controlling exogenous ligand binding. In this work, we investigated the influence of the Cys46/Cys55 disulfide bond on the redox properties and on the pH-dependent conformational equilibria of hNgb, using Uv-vis spectroelectrochemistry, cyclic voltammetry, electronic absorption spectroscopy and magnetic circular dichroism (MCD). We found that the S-S bridge significantly affects the heme Fe(III) to Fe(II) reduction enthalpy (deltaH°’rc) and entropy (deltaS°’rc), mostly as a consequence of changes in the reduction-induced solvent reorganization effects, without affecting the axial ligand-binding interactions and the polarity and electrostatics of the heme environment. Between pH 3 and 12, the electronic properties of the heme of ferric hNgb are sensitive to five acid-base equilibria, which are scarcely affected by the Cys46/Cys55 disulfide bridge. The equilibria occurring at extreme pH values induce heme release, while those occurring between pH 5 and 10 alter the electronic properties of the heme without modifying its axial coordination and low spin state. They involve the sidechains of non-coordinating aminoacids close to the heme and at least one heme propionate.
2017
- Computational evidence support the hypothesis of neuroglobin also acting as an electron transfer species
[Articolo su rivista]
Paltrinieri, Licia; DI ROCCO, Giulia; Battistuzzi, Gianantonio; Borsari, Marco; Sola, Marco; Ranieri, Antonio; Zanetti Polzi, Laura; Daidone, Isabella; Bortolotti, Carlo Augusto
abstract
Neuroglobin (Ngb) is a recently identified hexa-coordinated globin, expressed in the nervous system of humans. Its physiological role is still debated: one hypothesis is that Ngb serves as an electron transfer (ET) species, possibly by reducing cytochrome c and preventing it to initiate the apoptotic cascade. Here, we use the perturbed matrix method (PMM), a mixed quantum mechanics/molecular dynamics approach, to investigate the redox thermodynamics of two neuroglobins, namely the human Ngb and GLB-6 from invertebrate Caenorhabditis elegans. In particular, we calculate the reduction potential of the two globins, resulting in an excellent agreement with the experimental values, and we predict the reorganization energies, λ, which have not been determined experimentally yet. The calculated λ values match well those reported for known ET proteins and thereby support a potential involvement in vivo of the two globins in ET processes.
2017
- Computational investigation of the electron transfer complex between neuroglobin and cytochrome c
[Articolo su rivista]
Zanetti Polzi, Laura; Battistuzzi, Gianantonio; Borsari, Marco; Pignataro, Marcello; Pltrinieri, Licia Paltrinieri; Daidone, Isabella; Bortolotti, Carlo Augusto
abstract
Neuroglobin (Ngb) was the first vertebrate nerve globin to be identified. Since then, different physiological roles have been hypothesised for this hexa-coordinated globin, but its function is far from being unambiguously assigned. In a previous work, we collected first evidences of Ngb potentially taking part to electron transfer (ET) processes in vivo, investigating the redox thermodynamics of this globin. Here, we perform a computational investigation on the complex between Ngb and its putative in vivo partner cyt c and on the ET process between the two species. The simulated structure of the complex is amenable for ET in terms of distance and relative protein orientation. Moreover, the redox-dependent stability of the predicted Ngb-cyt c adduct and the very good agreement between calculated determinants to the ET rate and those of paradigmatic metalloproteins acting as electron shuttles all support a potential role of neuroglobin as an electron transfer species.
2017
- Corrigendum: Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity
[Articolo su rivista]
Bernini, F.; Malferrari, D.; Pignataro, M.; Bortolotti, C. A.; Di Rocco, G.; Lancellotti, L.; Kayed, R.; Borsari, M.; Del Monte, F.; Castellini, E.; Brigatti, M. F.
abstract
2017
- Label-free detection of interleukin-6 using electrolyte gated organic field effect transistors
[Articolo su rivista]
Diacci, Chiara; Berto, Marcello; Di Lauro, Michele; Bianchini, Elena; Pinti, Marcello; Simon, Daniel T.; Biscarini, Fabio; Bortolotti, Carlo A.
abstract
Cytokines are small proteins that play fundamental roles in inflammatory processes in the human body. In particular, interleukin (IL)-6 is a multifunctional cytokine, whose increased levels are associated with infection, cancer, and inflammation. The quantification of IL-6 is therefore of primary importance in early stages of inflammation and in chronic diseases, but standard techniques are expensive, time-consuming, and usually rely on fluorescent or radioactive labels. Organic electronic devices and, in particular, organic field-effect transistors (OFETs) have been proposed in the recent years as novel platforms for label-free protein detection, exploiting as sensing unit surface-immobilized antibodies or aptamers. Here, the authors report two electrolyte-gated OFETs biosensors for IL-6 detection, featuring monoclonal antibodies and peptide aptamers adsorbed at the gate. Both strategies yield biosensors that can work on a wide range of IL-6 concentrations and exhibit a remarkable limit of detection of 1 pM. Eventually, electrolyte gated OFETs responses have been used to extract and compare the binding thermodynamics between the sensing moiety, immobilized at the gate electrode, and IL-6.
2017
- Liquid-Gated Organic Electronic Devices Based on High-Performance Solution-Processed Molecular Semiconductor
[Articolo su rivista]
DI LAURO, Michele; Berto, Marcello; Giordani, Martina; Benaglia, Simone; Schweicher, Guillaume; Vuillaume, Dominique; Bortolotti, Carlo Augusto; Geerts, Yves H.; Biscarini, Fabio
abstract
High-mobility organic semiconductors such as [1]benzothieno[3,2-b]benzothiophene (BTBT) derivatives are potential candidates for ultrasensitive biosensors. Here 2,7-dioctyl BTBT (C8-BTBT-C8)-based liquid-gated organic electronic devices are demonstrated with two device architectures, viz. electrolyte-gated organic field-effect transistor (EGOFET) and electrolyte-gated organic synapstor (EGOS), and different electrode materials, viz. gold and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS). EGOFETs exhibit a mean transconductance of about 45 µS, on a par with literature, and a max value up to 256 µS at the state-of-the-art in aqueous electrolyte, with a mean product of charge mobility and effective capacitance of about 0.112 and 0.044 µS V−1 for gold and PEDOT:PSS electrodes, respectively. EGOSs exhibit a dynamic response with 15 ms characteristic timescale with Au electrodes and about twice with PEDOT:PSS electrodes. These results demonstrate a promising route for sensing applications in physiological environment based on fully solution-processed whole-organic electronic devices featuring ultrahigh sensitivity and fast response.
2017
- Self-assembled monolayers in organic electronics
[Articolo su rivista]
Casalini, Stefano; Bortolotti, Carlo Augusto; Leonardi, Francesca; Biscarini, Fabio
abstract
Self-assembly is possibly the most effective and versatile strategy for surface functionalization. Self-assembled monolayers (SAMs) can be formed on (semi-)conductor and dielectric surfaces, and have been used in a variety of technological applications. This work aims to review the strategy behind the design and use of self-assembled monolayers in organic electronics, discuss the mechanism of interaction of SAMs in a microscopic device, and highlight the applications emerging from the integration of SAMs in an organic device. The possibility of performing surface chemistry tailoring with SAMs constitutes a versatile approach towards the tuning of the electronic and morphological properties of the interfaces relevant to the response of an organic electronic device. Functionalisation with SAMs is important not only for imparting stability to the device or enhancing its performance, as sought at the early stages of development of this field. SAM-functionalised organic devices give rise to completely new types of behavior that open unprecedented applications, such as ultra-sensitive label-free biosensors and SAM/organic transistors that can be used as robust experimental gauges for studying charge tunneling across SAMs.
2017
- Specific Dopamine Sensing Based on Short-Term Plasticity Behavior of a Whole Organic Artificial Synapse
[Articolo su rivista]
Giordani, Martina; Berto, Marcello; Di Lauro, Michele; Bortolotti, Carlo A.; Zoli, Michele; Biscarini, Fabio
abstract
In this work, we demonstrate the ultrasensitive and selective detection of dopamine by means of a neuro-inspired device platform without the need of a specific recognition moiety. The sensor is a whole organic device featuring two electrodes made of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate - PEDOT:PSS - patterned on a polydymethylsiloxane - PDMS - flexible substrate. One electrode is pulsed with a train of voltage square waves, to mimic the presynaptic neuron behavior, while the other is used to record the displacement current, mimicking the postsynaptic neuron. The current response exhibits the features of synaptic Short-Term Plasticity (STP) with facilitating or depressing response according to the stimulus frequency. We found that the response characteristic time USTPdepends on dopamine (DA) concentration in solution. The dose curve exhibits superexponential sensitivity at the lowest concentrations below 1 nM. The sensor detects [DA] down to 1 pM range. We assess the sensor also in the presence of ascorbic acid (AA) and uric acid (UA). Our sensor does not respond to UA, but responds to AA only at concentration above 100 μM. However, it is still able to detect DA down to 1 pM range in the presence of [AA] = 100 μM and 100 pM in the presence of [UA] = 3 μM, these values for AA and UA being the physiological levels in the cerebrospinal fluid and the striatum, respectively.
2016
- Biorecognition in Organic Field Effect Transistors Biosensors: The Role of the Density of States of the Organic Semiconductor
[Articolo su rivista]
Berto, Marcello; Casalini, Stefano; Di Lauro, Michele; Marasso, Simone L; Cocuzza, Matteo; Perrone, Denis; Pinti, Marcello; Cossarizza, Andrea; Pirri, Candido F; Simon, Daniel T; Berggren, Magnus; Zerbetto, Francesco; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract
Biorecognition is a central event in biological processes in the living systems that is also widely exploited in technological and health applications. We demonstrate that the Electrolyte Gated Organic Field Effect Transistor (EGOFET) is an ultrasensitive and specific device that allows us to quantitatively assess the thermodynamics of biomolecular recognition between a human antibody and its antigen, namely, the inflammatory cytokine TNFα at the solid/liquid interface. The EGOFET biosensor exhibits a superexponential response at TNFα concentration below 1 nM with a minimum detection level of 100 pM. The sensitivity of the device depends on the analyte concentration, reaching a maximum in the range of clinically relevant TNFα concentrations when the EGOFET is operated in the subthreshold regime. At concentrations greater than 1 nM the response scales linearly with the concentration. The sensitivity and the dynamic range are both modulated by the gate voltage. These results are explained by establishing the correlation between the sensitivity and the density of states (DOS) of the organic semiconductor. Then, the superexponential response arises from the energy-dependence of the tail of the DOS of the HOMO level. From the gate voltage-dependent response, we extract the binding constant, as well as the changes of the surface charge and the effective capacitance accompanying biorecognition at the electrode surface. Finally, we demonstrate the detection of TNFα in human-plasma derived samples as an example for point-of-care application.
2016
- Emerging role of Lon protease as a master regulator of mitochondrial functions
[Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; Nasi, Milena; De Biasi, Sara; Bortolotti, Carlo Augusto; Iannone, Anna; Cossarizza, Andrea
abstract
Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
2016
- Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes
[Articolo su rivista]
DI ROCCO, Giulia; Bernini, Fabrizio; Borsari, Marco; Martinelli, Ilaria; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Ranieri, Antonio; Caselli, Monica; Sola, Marco; Ponterini, Glauco
abstract
The intrinsic fluorescence of recombinant proteins offers a powerful tool to detect and characterize structural changes induced by chemical or biological stimuli. We show that metal-ion binding to a hexahistidine tail can significantly broaden the range of such structurally sensitive fluorescence observables. Bipositive metal-ions as Cu2+, Ni2+ and Zn2+ bind 6xHis-tag azurin and its 6xHis-tagged R129W and W48A-R129W mutants with good efficiency and, thereby, quench their intrinsic fluorescence. Due to a much more favourable spectral overlap, the 6xHis-tag/Cu2+ complex(es) are the most efficient quenchers of both W48 and W129 emissions. Based on simple Förster-type dependence of energy-transfer efficiency on donor/acceptor distance, we can trace several excitation-energy transfer paths across the protein structure. Unexpected lifetime components in the azurin 6xHis-tag/Cu2+ complex emission decays reveal underneath complexity in the conformational landscape of these systems. The new tryptophan emission quenching paths provide additional signals for detecting and identifying protein structural changes.
2016
- Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity
[Articolo su rivista]
Bernini, Fabrizio; Malferrari, Daniele; Pignataro, Marcello; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Lancellotti, Lidia; Brigatti, Maria Franca; Kayed, Rakez; Borsari, Marco; Del Monte, Federica; Castellini, Elena
abstract
The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology.
2016
- The dynamics of the β-propeller domain in Kelch protein KLHL40 changes upon nemaline myopathy-associated mutation
[Articolo su rivista]
Lambrughi, Matteo; Lucchini, Matteo; Pignataro, Marcello; Sola, Marco; Bortolotti, Carlo Augusto
abstract
Evolutionarily widespread, functionally and structurally diverse and still largely unexplored, Kelch proteins, characterized by the presence of a conserved C-terminal β-propeller, are implicated in a number of diverse fundamental biological functions, including cytoskeletal arrangement, regulation of cell morphology and organization, and protein degradation. Mutations in the genes encoding for Kelch superfamily members are being discovered as the cause of several neuromuscular diseases and cancer. The E528K mutation in Kelch protein KLHL40, which regulates skeletal muscle myogenesis, has been identified as a frequent cause of severe autosomal-recessive nemaline myopathy (NM). We use all-atom molecular dynamics simulations to characterize the dynamic behaviour of the β-propeller of the wild-type protein and identify correlated motions underlying the in vivo functionality. We also modelled the NM-associated mutation and we found that it does not lead to dramatic disruption of the β-propeller architecture; yet, residue 528 is a hub in the correlated motions of the domain, and mutation-induced local structural alterations are propagated to the whole protein, affecting its dynamics and physicochemical properties, which are fundamental for in vivo interaction with partners. Our results indicate that rational design of drugs can be envisioned as a strategy for restoring the internal network of communication and resetting KLHL40 to its physiological state.
2016
- The substrate is a pH-controlled second gate of electrolyte-gated organic field-effect transistor
[Articolo su rivista]
Di Lauro, Michele; Casalini, Stefano; Berto, Marcello; Campana, Alessandra; Cramer, Tobias; Murgia, Mauro; Geoghegan, Mark Anthony; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract
Electrolyte-gated organic field-effect transistors (EGOFETs), based on ultrathin pentacene films on quartz, were operated with electrolyte solutions whose pH was systematically changed. Transistor parameters exhibit nonmonotonic variation versus pH, which cannot be accounted for by capacitive coupling through the Debye-Helmholtz layer. The data were fitted with an analytical model of the accumulated charge in the EGOFET, where Langmuir adsorption was introduced to describe the pH-dependent charge buildup at the quartz surface. The model provides an excellent fit to the threshold voltage and transfer characteristics as a function of the pH, which demonstrates that quartz acts as a second gate controlled by pH and is mostly effective from neutral to alkaline pH. The effective capacitance of the device is always greater than the capacitance of the electrolyte, thus highlighting the role of the substrate as an important active element for amplification of the transistor response.
2016
- Whole organic electronic synapses for dopamine detection
[Relazione in Atti di Convegno]
Giordani, Martina; DI LAURO, Michele; Berto, Marcello; Bortolotti, Carlo Augusto; Vuillaume, Dominique; Gomes, Henrique L.; Zoli, Michele; Biscarini, Fabio
abstract
A whole organic artificial synapse has been fabricated by patterning PEDOT:PSS electrodes on PDMS that are biased in frequency to yield a STP response. The timescale of the STP response is shown to be sensitive to the concentration of dopamine, DA, a neurotransmitter relevant for monitoring the development of Parkinson's disease and potential locoregional therapies. The sensitivity of the sensor towards DA has been validated comparing signal variation in the presence of DA and its principal interfering agent, ascorbic acid, AA. The whole organic synapse is biocompatible, soft and flexible, and is attractive for implantable devices aimed to real-time monitoring of DA concentration in bodily fluids. This may open applications in chronic neurodegenerative diseases such as Parkinson's disease.
2015
- A few key residues determine the high redox potential shift in azurin mutants
[Articolo su rivista]
Zanetti Polzi, Laura; Bortolotti, Carlo Augusto; Daidone, Isabella; Aschi, Massimiliano; Amadei, Andrea; Corni, Stefano
abstract
The wide range of variability of the reduction potential (E(0)) of blue-copper proteins has been the subject of a large number of studies in the past several years. In particular, a series of azurin mutants have been recently rationally designed tuning E(0) over a very broad range (700 mV) without significantly altering the redox-active site [Marshall et al., Nature, 2009, 462, 113]. This clearly suggests that interactions outside the primary coordination sphere are relevant to determine E(0) in cupredoxins. However, the molecular determinants of the redox potential variability are still undisclosed. Here, by means of atomistic molecular dynamics simulations and hybrid quantum/classical calculations, the mechanisms that determine the E(0) shift of two azurin mutants with high potential shifts are unravelled. The reduction potentials of native azurin and of the mutants are calculated obtaining results in good agreement with the experiments. The analysis of the simulations reveals that only a small number of residues (including non-mutated ones) are relevant in determining the experimentally observed E(0) variation via site-specific, but diverse, mechanisms. These findings open the path to the rational design of new azurin mutants with different E(0).
2015
- Electrowetting of nitro-functionalized oligoarylene thiols self-assembled on polycrystalline gold
[Articolo su rivista]
Casalini, Stefano; Berto, Marcello; Bortolotti, Carlo Augusto; Foschi, Giulia; Operamolla, Alessandra; DI LAURO, Michele; Omar, Omar Hassan; Liscio, Andrea; Pasquali, Luca; Montecchi, Monica; Farinola, Gianluca M; Borsari, Marco
abstract
Four linear terarylene molecules (i) 4-nitro-terphenyl-4″-methanethiol (NTM), (ii) 4-nitro-terphenyl-3″,5″-dimethanethiol (NTD), (iii) ([1,1';4',1″] terphenyl-3,5-diyl)methanethiol (TM), and (iv) ([1,1';4',1″] terphenyl-3,5-diyl)dimethanethiol (TD) have been synthesized and their self-assembled monolayers (SAMs) have been obtained on polycrystalline gold. NTM and NTD SAMs have been characterized by X-ray photoelectron spectroscopy, Kelvin probe measurements, electrochemistry, and contact angle measurements. The terminal nitro group (-NO2) is irreversibly reduced to hydroxylamine (-NHOH), which can be reversibly turned into nitroso group (-NO). The direct comparison between NTM/NTD and TM/TD SAMs unambiguously shows the crucial influence of the nitro group on electrowetting properties of polycrystalline Au. The higher grade of surface tension related to NHOH has been successfully exploited for basic operations of digital μ-fluidics, such as droplets motion and merging.
2015
- Human Cofilin2: Towards the Comprehension of the Molecular Mechanism
[Abstract in Atti di Convegno]
DI ROCCO, Giulia; Pignataro, Marcello; Bortolotti, Carlo Augusto; Castellini, Elena; Lancellotti, Lidia; Borsari, Marco; Sola, Marco; Del Monte, F.
abstract
Cofilin is an evolutionarily highly conserved protein which belongs to the ADF/cofilin
family involved in the regulation of actin-filament dynamics depolymerizing and/or
severing actin filaments. Phosphorylation on serine 3 inactivates cofilin [1,2] by
generation of a charge repulsion between cofilin and actin, which is thought to occur
without altering the protein structure [3]. In terms of physiological functions, cofilin-
2 is the least understood member of this protein family, which is present
predominantly in skeletal and cardiac muscle [4-6]. In reducing media, even
phosphatidylinositol 4,5-bisphosphate-bound cofilin is active, leading to actin
dynamics in the vicinity of the plasma membrane. This mechanism has been
proposed to explain why dendritic cells that are able to increase the thiol pool in
antigen-specific T cells enable T cell activation even under oxidative stress
conditions. On the contrary, cofilin is inactivated by oxidation, provoking T-cell
hyporesponsiveness or necrotic-like programmed cell death [7]. In this study we
present the production, the physico-chemical characterization and the modelled
structure of the wt and the phosphorylated-mimicking S3D variant of the human
cofilin2. The study allowed the evaluation of the structural differences between the
active and the inactive protein while an electrochemical and fluorometric approach
provided new data to increase in the understanding of the cofilin-action mechanism.
1. Agnew BJ, Minamide LS, Bamburg JR. J Biol Chem 1995; 270:17582–17587.
2. Moriyama K, Iida K, Yahara I. Genes Cells 1996; 1:73–86.
3. Blanchoin L, Robinson RC, Choe S, Pollard TD. J Mol Biol 2000;295:203–211.
4. Bernstein BW, Bamburg JR. Trends Cell Biol 2010;20(4):187–95.
5. Agrawal PB, Joshi M, Savic Tetal.. Hum Mol Genet. 2012 May 15; 21(10): 2341–2356
6. C. Thirion et al. (Eur. J. Biochem. 268)-2001
7. Y. Samstag, I. John, G. H. Wabnitz Immunological Reviews 256 (2013) 30-47.
2015
- Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen
[Articolo su rivista]
Ranieri, Antonio; Millo, D.; Di Rocco, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Sola, Marco
abstract
Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc–CL) immobilized at IMM. In this work, we have studied electrochemically cytc–CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc–CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc–CL in solution. In particular, immobilized cytc–CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/− coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH– states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc–CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade.
2015
- Multiscale sensing of antibody-antigen interactions by organic transistors and single-molecule force spectroscopy
[Articolo su rivista]
Casalini, Stefano; Dumitru, Andra C.; Leonardi, Francesca; Bortolotti, Carlo Augusto; Herruzo, Elena T.; Campana, Alessandra; De Oliveira, Rafael F.; Cramer, Tobias; Garcia, Ricardo; Biscarini, Fabio
abstract
Antibody-antigen (Ab-Ag) recognition is the primary event at the basis of many biosensing platforms. In label-free biosensors, these events occurring at solid-liquid interfaces are complex and often difficult to control technologically across the smallest length scales down to the molecular scale. Here a molecular-scale technique, such as single-molecule force spectroscopy, is performed across areas of a real electrode functionalized for the immunodetection of an inflammatory cytokine, viz. interleukin-4 (IL4). The statistical analysis of force-distance curves allows us to quantify the probability, the characteristic length scales, the adhesion energy, and the time scales of specific recognition. These results enable us to rationalize the response of an electrolyte-gated organic field-effect transistor (EGOFET) operated as an IL4 immunosensor. Two different strategies for the immobilization of IL4 antibodies on the Au gate electrode have been compared: antibodies are bound to (i) a smooth film of His-tagged protein G (PG)/Au; (ii) a 6-aminohexanethiol (HSC6NH2) self-assembled monolayer on Au through glutaraldehyde. The most sensitive EGOFET (concentration minimum detection level down to 5 nM of IL4) is obtained with the first functionalization strategy. This result is correlated to the highest probability (30%) of specific binding events detected by force spectroscopy on Ab/PG/Au electrodes, compared to 10% probability on electrodes with the second functionalization. Specifically, this demonstrates that Ab/PG/Au yields the largest areal density of oriented antibodies available for recognition. More in general, this work shows that specific recognition events in multiscale biosensors can be assessed, quantified, and optimized by means of a nanoscale technique.
2015
- Surface Immobilized His-tagged Azurin as a Model Interface for the Investigation of Vectorial Electron Transfer in Biological Systems
[Articolo su rivista]
Casalini, Stefano; Berto, Marcello; Kovtun, Alessandro; Operamolla, Alessandra; DI ROCCO, Giulia; Facci, Paolo; Liscio, Andrea; Farinola, Gianluca M.; Borsari, Marco; Bortolotti, Carlo Augusto
abstract
A model system for the electrochemical investigation of vectorial electron transfer in biological systems was designed, assembled and characterized. Gold electrodes, functionalized with a -OCH3 terminated, aromatic self-assembled monolayer, were used as a substrate for the adsorption of variants of copper- containing, redox metalloprotein azurin. The engineered azurin bears a polyhistidine tag at its C-terminus. Thanks to the presence of the solvent exposed tag, which chelates Cu2+ ions in solution, we introduced an exogenous redox centre. The different reduction potentials of the two redox centres and their positioning with respect to the surface are such that electron transfer from the exogenous copper centre and the electrode is mediated by the native azurin active site, closely paralleling electron transfer processes in naturally occurring multicentre metalloproteins
2015
- Thermodynamics and kinetics of reduction and species conversion at a hydrophobic surface for mitochondrial cytochromes c and their cardiolipin adducts
[Articolo su rivista]
Ranieri, Antonio; DI ROCCO, Giulia; Millo, Diego; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Lancellotti, Lidia; Borsari, Marco; Sola, Marco
abstract
Cytochrome c(cytc) and its adduct with cardiolipin (CL) were immobilized on a hydrophobic SAM-coated electrode surface yielding a construct which mimics the environment experienced by the complex at the inner mitochondrial membrane where it plays a role in cell apoptosis. Under these conditions, both species undergo an equilibrium between a six-coordinated His/His-ligated and a five-coordinated His/- ligated forms stable in the oxidized and in the reduced state, respectively. The thermodynamics of the oxidation-state dependent species conversion were determined by temperature-dependent diffusionless voltammetry experiments. CL binding stabilizes the immobilized reduced His/- ligated form of cytc which was found previously to catalytically reduce dioxygen. Here, this adduct is also found to show pseudoperoxidase activity, catalysing reduction of hydrogen peroxide. These effects would impart CL with an additional role in the cytc-mediated peroxidation leading to programmed cell death. Moreover, Immobilized cytc exchanges electrons more slowly upon CL binding possibly due to changes in solvent reorganization effects at the protein-SAM interface.
2014
- Effect of motional restriction on the unfolding properties of a cytochrome c featuring a His/Met-His/His ligation switch
[Articolo su rivista]
Ranieri, Antonio; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Borsari, Marco; Paltrinieri, Licia; DI ROCCO, Giulia; Sola, Marco
abstract
The K72A/K73H/K79A variant of cytochrome c undergoes a reversible change from a His/Met to a His/His axial heme ligation upon urea-induced unfolding slightly below neutral pH. The unfolded form displays a dramatically lower reduction potential than the folded species along with a pseudo-peroxidase activity. We have studied electrochemically the effects of urea-induced unfolding on the protein electrostatically immobilized on an electrode surface functionalized by means of a negatively charged molecular spacer. The latter mimics the electrostatic interaction with the inner mitochondrial membrane. This behavior has been compared with the unfolding of the same species in solution. This system constitutes a model to decipher the role of the above electrostatic interaction in the unfolding of cytochrome c at physiological pH upon interaction with the membrane component phospholipid cardiolipin in the early stages of the apoptosis cascade. We found that immobilization obstacles protein unfolding due to structural constraints at the interface imposed by protein-SAM interaction.
2014
- How the Reorganization Free Energy Affects the Reduction Potential of Structurally Homologous Cytochromes
[Articolo su rivista]
Isabella, Daidone; Andrea, Amadei; Francesco, Zaccanti; Borsari, Marco; Bortolotti, Carlo Augusto
abstract
Differences in the reduction potential E0 among structurally similar metalloproteins cannot always be fully explained on the basis of their 3-D structures. We investigate the molecular determinants to E0 using the mixed quantum mechanics/molecular mechanics approach named perturbed matrix method (PMM); after comparison with experimental values to assess the reliability of our calculations, we investigate the relationship between the change in free energy upon reduction ΔA0 and the reorganization energy. We find that the reduction potential of cytochromes can be regarded as the result of the sum of two terms, one being mostly dependent on the energy fluctuations within a limited range around the mean transition energy and the second being mostly dependent linearly on the difference Δλ = λred – λox of the reorganization free energies for the ox → red (λred) and for the red → ox (λox) relaxations.
2014
- Influence of the dynamic interplay between protein and solvent on the redox properties of blue copper proteins
[Abstract in Rivista]
Battistuzzi, Gianantonio; Paltrinieri, Licia; Borsari, Marco; Bortolotti, Carlo Augusto; Sola, Marco; C., Dennison; Corni, Stefano
abstract
We used direct electrochemistry and MD simulations to investigate the redox reactivity of native azurin and four chimeric cupredoxins, in which the ligand-containing loop of azurin has been replaced either with that of other members of the blue copper family or with synthetic sequences featuring only Ala residues. It turns out that the dynamic interplay between protein and solvent is the key factor determining the redox properties of these hallmark ET systems. In particular, the dynamics of the small, metal-binding loop region controls the outer-sphere reorganization energy. The molecular determinants to the reduction potential were also investigated. Moreover, the dynamics between the protein scaffold and the surrounding solvent proved to be crucial in determining the pKa of the protonation of the C-terminal copper binding His in the reduced proteins.
2014
- Solid-state electron transport via cytochrome c depends on electronic coupling to electrodes and across the protein
[Articolo su rivista]
N., Amdursky; D., Ferber; Bortolotti, Carlo Augusto; D. A., Dolgikh; R. V., Chertkova; I., Pecht; M., Sheves; D., Cahen
abstract
Electronic coupling to electrodes, Γ, as well as that across the
examined molecules, H, is critical for solid-state electron transport (ETp) across proteins.
Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron mediating protein cytochrome c (CytC), measured in a mono-
layer configuration. Currents via CytC are temperature-independent between 30 and∼130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein–electrode binding significantly increases
Γ, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than
those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy,
Ea, of the ETp by more
than a factor of two. The importance of H
was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode–protein(–
heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower
Ea values (from high-temperature
data) and higher conductance at low temperatures (in the temperature-independent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width)
2014
- Surface Packing Determines the Redox Potential Shift of Cytochrome c Adsorbed on Gold
[Articolo su rivista]
Laura Zanetti, Polzi; Isabella, Daidone; Bortolotti, Carlo Augusto; Stefano, Corni
abstract
Thermodynamic and dynamic properties of iso-1-cytochrome c covalently bound to a bare gold surface are here investigated by large scale atomistic simulations. The reduction potential of the protein for low and high surface concentrations is calculated showing a good agreement with experimental estimates. The origin of the dependence of the reduction potential on the surface concentration is investigated and is demonstrated to stem from the changing polarizability of the environment surrounding the protein, a mechanism reminiscent of crowding effects. Moreover, structural analyses are performed revealing relevant changes induced by the presence of the electrode on the dynamic properties of cytochrome c. In particular, one of the two cavities previously identified on the protein surface [Bortolotti et al. J. Am. Chem. Soc., 2012, 134, 13670], and that reversibly open in cytochrome c freely diffusing in solution, is found to be deformed when the protein is adsorbed on gold. This modification exemplifies a mechanism that potentially leads to changes in the protein properties by surface-induced modification of its dynamical behavior.
2014
- Unambiguous Assignment of Reduction Potentials in Diheme Cytochromes
[Articolo su rivista]
I., Daidone; Paltrinieri, Licia; A., Amadei; Battistuzzi, Gianantonio; Sola, Marco; Borsari, Marco; Bortolotti, Carlo Augusto
abstract
Perturbed matrix method calculations are performed on a diheme cytochrome c (DHC) protein, in order to assign previously experimentally detemined reduction potentials (E°) to their corresponding heme groups. Very good agreement between calculated values to experimental data prove that the present approach can be used as a predictive tool of redox thermodynamic properties of multicenter redox proteins in the absence of experimental data, or in synergy with state-of-the art spectroscopic and electrochemical approaches to obtain a detailed picture of electron transfer processes within these complex systems.
2013
- A general statistical mechanical approach for modeling redox thermodynamics: the reaction and reorganization free energies
[Articolo su rivista]
Andrea, Amadei; Isabella, Daidone; Bortolotti, Carlo Augusto
abstract
By using rigorous statistical mechanical derivations we obtain a general theoretical model providing the thermodynamics of redox processes, with a focus on the reaction and reorganization free energies and on the relationship between these key thermodynamic quantities. In particular, we define two distinct reorganization free energies, lP and lR, for the reactants (R) A products (P) reaction and for the inverse process, respectively. We first derive in principle exact relationships, then gradually introduce different levels of approximation to obtain more and more simplified, though less general, working equations. The results of the calculation of thermodynamic properties for two model systems are then used to compare general and more approximated expressions and critically assess their applicability to the description of redox processes. Finally, we obtain specific relationships that can be used as a diagnostic tool to test the actual reliability of the assumption of Gaussian fluctuations, a priori accepted within Marcus theory, for any redox system under investigation. For both benchmark molecules studied in the present paper, the Gaussian approximation turns out to be inappropriate to describe the redox thermodynamics
2013
- Axial iron coordination and spin state change in a heme c upon electrostatic protein–SAM interaction
[Articolo su rivista]
DI ROCCO, Giulia; Ranieri, Antonio; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Alois, Bonifacio; Valter, Sergo; Borsari, Marco; Sola, Marco
abstract
A bacterial di-heme cytochrome c binds electrostatically to a gold electrode surface coated with a negatively charged COOH-terminated SAM adopting a sort of 'perpendicular' orientation. Cyclic voltammetry, Resonance Raman and SERRS spectroscopies indicate that the high-potential C-terminal heme center proximal to the SAM's surface undergoes an adsorption-induced swapping of one axial His ligand with a water molecule, which is probably lost in the reduced form, and a low- to high-spin transition. This coordination change for a bis-His ligated heme center upon an electrostatically-driven molecular recognition is as yet unprecedented, as well as the resulting increase in reduction potential. We discuss it in comparison with the known methionine ligand lability in monoheme cytochromes c occurring upon interaction with charged molecular patches. One possible implication of this finding in biological ET is that mobile redox partners do not behave as rigid and invariant bodies, but in the ET complex are subjected to molecular changes and structural fluctuations that affect in a complex way the thermodynamics and the kinetics of the process.
2013
- Enhancing Biocatalysis: The Case of Cytochrome c Unfolded Immobilized on Kaolinite
[Articolo su rivista]
Castellini, Elena; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Bernini, Fabrizio; Ranieri, Antonio
abstract
Urea-unfolded wild-type cytochrome c and its variants immobilized on kaolinite show peroxidase activity that is significantly higher than that of the folded wild-type protein.
The accessibility of the substrate to the metal center and the influence of strategic amino acidic residues on the surface of the protein are discussed. This approach sheds light on the factors affecting the catalytic activity of a new versatile biocatalytic interface.
2013
- How the Dynamics of the Metal-Binding Loop Region Controls the Acid Transition in Cupredoxins
[Articolo su rivista]
Paltrinieri, Licia; Borsari, Marco; Battistuzzi, Gianantonio; Sola, Marco; Christopher, Dennison; Bert L., de Groot; Stefano, Corni; Bortolotti, Carlo Augusto
abstract
Many reduced cupredoxins undergo a pH-dependent structural
rearrangement, triggered by protonation of the His ligand belonging to the C-terminal hydrophobic loop, usually termed the acid transition. At variance with several members of the cupredoxin family, the acid transition is not observed for azurin (AZ). We have addressed this issue by performing molecular dynamics simulations of AZ and four mutants, in which the C-terminal loop has been replaced with those of other cupredoxins or with polyalanine loops. All of the loop mutants undergo the acid transition in the pH range of 4.4−5.5. The main
differences between AZ and its loop mutants are the average value of the active site solvent accessible surface area and the extent of its fluctuations with time, together with an altered structure of the water layer around the copper center. Using functional mode analysis, we found that these variations arise from changes in nonbonding interactions in the second coordination sphere of the copper center, resulting from the loop mutation. Our results strengthen the view that the dynamics at the site relevant for function and its surroundings are crucial for protein activity and for metal-containing electron transferases.
2013
- Self-Assembly of Mono- And Bidentate Oligoarylene Thiols onto Polycrystalline Au
[Articolo su rivista]
Casalini, S.; Berto, M.; Leonardi, F.; Operamolla, A.; Bortolotti, Carlo Augusto; Borsari, Marco; Sun, W.; DI FELICE, Rosa; Corni, Stefano; Albonetti, C.; Hassan Omar, O.; Farinola, G. M.; Biscarini, Fabio
abstract
Four thiolated oligoarylene molecules (i) 4-methoxy-terphenyl-4″-methanethiol (MTM), (ii) 4-methoxy-terphenyl-3″,5″-dimethanethiol (MTD), (iii) 4-nitro-terphenyl-4″-methanethiol (NTM), and (iv) 4-nitro-terphenyl-3″,5″-dimethanethiol (NTD) were synthesized and self-assembled as monolayers (SAMs) on polycrystalline Au electrodes of organic field-effect transistors (OFETs). SAMs were characterized by contact angle and AC/DC electrochemical measurements, whereas atomic force microscopy was used for imaging the pentacene films grown on the coated electrodes. The electrical properties of functionalized OFETs, the electrochemical SAMs features and the morphology of pentacene films were correlated to the molecular organization of the thiolated oligoarylenes on Au, as calculated by means of the density functional theory. This multi-methodological approach allows us to associate the systematic replacement of the SAM anchoring head group (viz. methanethiol and dimethanethiol) and/or terminal tail group (viz. nitro-, -NO2, and methoxy, -OCH3) with the change of the electrical features. The dimethanethiol head group endows SAMs with higher resistive features along with higher surface tensions compared with methanethiol. Furthermore, the different number of thiolated heads affects the kinetics of Au passivation as well as the pentacene morphology. On the other hand, the nitro group confers further distinctive properties, such as the positive shift of both threshold and critical voltages of OFETs with respect to the methoxy one. The latter experimental evidence arise from its electron-withdrawing capability, which has been verified by both DFT calculations and DC electrochemical measurements.
2013
- The Active Site Loop Modulates the Reorganization Energy of Blue Copper Proteins by Controlling the Dynamic Interplay with Solvent
[Articolo su rivista]
Paltrinieri, Licia; Borsari, Marco; Ranieri, Antonio; Battistuzzi, Gianantonio; S., Corni; Bortolotti, Carlo Augusto
abstract
Understanding the factors governing the rate of electron transfer processes in proteins is crucial not only to a deeper understanding of redox processes in living organisms but also for the design of efficient devices featuring biological molecules. Here, molecular dynamics simulations performed on native azurin and four chimeric cupredoxins allow for the calculation of the reorganization energy and of structure-related quantities that were used to clarify the molecular determinants to the dynamics/function relationship in blue copper proteins. We find that the dynamics of the small, metal-binding loop region controls the outer-sphere reorganization energy not only by determining the exposure of the active site to solvent but also through the modulation of the redox-dependent rearrangement of the whole protein scaffold and of the surrounding water molecules.
2013
- Voltammetry of the cytochrome c-cardiolipin complex in the immobilized state. Implications in apoptosis initiation
[Abstract in Rivista]
DI ROCCO, Giulia; Ranieri, Antonio; Bortolotti, Carlo Augusto; Borsari, Marco; Battistuzzi, Gianantonio; Sola, Marco
abstract
A voltammetric behavior of the complex cytochrome c -Cardiolipin adsorbed on modified gold electrodes has been described
2012
- A Bis-Histidine-Ligated Unfolded Cytochrome c Immobilized on Anionic SAM Shows Pseudo-Peroxidase Activity
[Articolo su rivista]
Ranieri, Antonio; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Monari, Stefano; Sola, Marco
abstract
Urea-unfolded yeast iso-1-cytochrome c electrostatically adsorbed on a gold electrode coated with an anionic self-assembled monolayer yields a heme-mediated electrocatalytic reduction of H2O2 (pseudo-peroxidase activity). Under the same conditions, native cytochrome c is inactive. In the unfolded protein, the Met80 heme iron ligand is replaced by a histidine residue yielding a bis-His-ligated form. H2O2 electrocatalysis occurs with an efficient mechanism likely involving direct H2O2 interaction with the iron(II) center and formation of a transient ferryl group. Comparison of the catalytic activity of a few urea-unfolded single and double Lys-to-Ala variants shows that the kinetic affinity of H2O2 for the heme iron and kcat of the bis-His-ligated form are strongly affected by the geometry of protein adsorption, controlled by specific surface lysine residues.
2012
- A surface-immobilized cytochrome c variant provides a pH-controlled molecular switch
[Articolo su rivista]
Bortolotti, Carlo Augusto; Paltrinieri, Licia; Monari, Stefano; Ranieri, Antonio; Borsari, Marco; Battistuzzi, Gianantonio; Sola, Marco
abstract
The K72A/K73H/K79A mutant of yeast iso-1-cytochrome c immobilized on a conductive substrate reversibly interconverts between the native-like, His-Met heme-ligated form and a His-His-ligated conformer with remarkably different redox and enzymatic properties. This transition is activated by changing the pH in a narrow range around neutrality.
2012
- Mono/bidentate thiol oligoarylene-based self-assembled monolayers (SAMs) for interface engineering
[Articolo su rivista]
S., Casalini; F., Leonardi; Bortolotti, Carlo Augusto; A., Operamolla; O. H., Omar; Paltrinieri, Licia; C., Albonetti; G. M., Farinola; Biscarini, Fabio
abstract
A new set of linear oligoarylene thiol molecules, namely (4′-(Thiophen-2-yl)Biphenyl-3,5-diyl)Dimethanethiol (TBD), (4′-(Thiophen-2-yl)Biphenyl-4-yl)Methanethiol (TBM) and ([1,1′;4′,1′′]Terphenyl-3,5-diyl)Dimethanethiol (TD), were synthesized and used for functionalizing the polycrystalline gold electrodes. Such molecules differ for the number of anchoring groups (TBM vs. TBD) and the terminal rings (TD vs. TBD). As shown by electrochemical measurements, they form homogeneous and pinholes-free self-assembly monolayers (SAMs) when deposited on the gold electrode. Moreover, the wettability of the functionalized surface and the morphological changes of pentacene films grown on SAMs were investigated by contact angle and atomic force microscopy, respectively. OTFT has been used as organic gauge for investigating the metal–SAM–organic semiconductor structure. Charge carriers mobility, threshold voltage, contact resistance were measured in both air and vacuum to assess the influence of the anchoring groups and the terminal rings to the transistor performance. Although these SAMs do not show an improvement of mobility due to an increase of contact resistance, they allow a better modulation of the current flowing across the electrode–organic semiconductor (OS) interface, pointing out the structural differences between the three SAMs in terms of resistance drop combined with the critical voltage.
2012
- pH and Solvent H/D Isotope Effects on the Thermodynamics
and Kinetics of Electron Transfer for Electrode-Immobilized
Native and Urea-Unfolded Stellacyanin
[Articolo su rivista]
Ranieri, Antonio; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Sola, Marco
abstract
The thermodynamics of Cu(II) to Cu(I) reduction and the kinetics of the electron transfer (ET) process for Rhus vernicifera stellacyanin (STC) immobilized on a decane-1-thiol coated gold
electrode have been measured through cyclic voltammetry at varying pH and temperature, in the presence of urea and in D2O. Immobilized STC undergoes a limited conformational change that
mainly results in an enhanced exposure of one or both copper binding histidines to solvent which slightly stabilizes the cupric state and increases histidine basicity. The large immobilization-induced increase in the pKa for the acid transition (from 4.5 to 6.3) makes this electrode-SAM-protein construct an attractive candidate as a biomolecular ET switch operating near neutral pH in molecular electronics. Such a potential interest is increased by the robustness of this interface against chemical unfolding as it undergoes only moderate changes in the reduction thermodynamics and in the ET rate in the presence of up to 8 M urea. The sensitivity of these parameters to solvent H/D isotope effects testifies the role of protein solvation as effector of the thermodynamics and kinetics of ET.
2012
- Role of Met80 and Tyr67 in the Low-pH Conformational Equilibria ofCytochrome c
[Articolo su rivista]
Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Bellei, Marzia; DI ROCCO, Giulia; J., Salewski; P., Hildebrandt; Sola, Marco
abstract
The low-pH conformational equilibria of ferricyeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, andM80A/Y67A variants were studied from pH 7 to 2 at low ionicstrength through electronic absorption, magnetic circulardichroism, and resonance Raman spectroscopies. For wild-typeycc, the protein structure, axial heme ligands, and spin state ofthe iron atom convert from the native folded His/Met low-spin(LS) form to a molten globule His/H2O high-spin (HS) formand a totally unfolded bis-aquo HS state, in a single cooperativetransition with an apparent pKa of ∼3.0. An analogouscooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pKa of ∼4.2, and by an equilibrium between a His/OH−-ligated LS and a His/H2O-ligated HS conformer, with a pKa of∼5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increasedstability of the molten globule state that is formed at higher pH values than the other species. These data show that removal ofthe axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pHconformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule andrenders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the threedimensionalstructure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues71−85 and 40−57.
2012
- The Met80Ala point mutation enhances the peroxidase activity of immobilized cytochrome c
[Articolo su rivista]
Ranieri, Antonio; Bernini, Fabrizio; Bortolotti, Carlo Augusto; Castellini, Elena
abstract
The effects of replacement of the axial methionine 80 hemeligand with a non-coordinating alanine on the peroxidaseactivity of kaolinite-immobilized cytochrome c wereinvestigated at different pH values. The catalytic activity10 of the adsorbed mutant was found remarkably higher thanthat of wild-type cytochrome c. The pH dependence ofVmax and KM values is discussed in term of accessibility ofthe substrates to the metal center and surface charge ofkaolinite. Our approach, based on the combined use of15 adsorption on kaolinite and protein engineering, endowsthis bioinorganic interface with remarkable catalyticproperties.
2012
- The Reversible Opening of Water Channels in Cytochrome c Modulates the Heme Iron Reduction Potential
[Articolo su rivista]
Bortolotti, Carlo Augusto; Amadei, A.; Aschi, M.; Borsari, Marco; Corni, S.; Sola, Marco; Daidone, I.
abstract
Dynamic protein–solvent interactions are fundamental for life processes, but their investigation is still experimentally very demanding. Molecular dynamics simulations up to hundreds of nanoseconds can bring to light unexpected events even for extensively studied biomolecules. This paper reports a combined computational/experimental approach that reveals the reversible opening of two distinct fluctuating cavities in Saccharomyces cerevisiae iso-1-cytochrome c. Both channels allow water access to the heme center. By means of a mixed quantum mechanics/molecular dynamics (QM/MD) theoretical approach, the perturbed matrix method (PMM), that allows to reach long simulation times, changes in the reduction potential of the heme Fe3+/Fe2+ couple induced by the opening of each cavity are calculated. Shifts of the reduction potential upon changes in the hydration of the heme propionates are observed. These variations are relatively small but significant and could therefore represent a tool developed by cytochrome c for the solvent driven, fine-tuning of its redox functionality.
2012
- Understanding the Mechanism of Short-Range Electron TransferUsing an Immobilized Cupredoxin
[Articolo su rivista]
Monari, Stefano; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; S., Yanagisawa; K., Sato; C., Li; I., Salard; D., Kostrz; Borsari, Marco; Ranieri, Antonio; C., Dennison; Sola, Marco
abstract
The hydrophobic patch of azurin (AZ)from Pseudomonas aeruginosa is an important recognitionsurface for electron transfer (ET) reactions. The influenceof changing the size of this region, by mutating the Cterminalcopper-binding loop, on the ET reactivity of AZadsorbed on gold electrodes modified with alkanethiol selfassembledmonolayers (SAMs) has been studied. Thedistance-dependence of ET kinetics measured by cyclicvoltammetry using SAMs of variable chain length,demonstrates that the activation barrier for short-rangeET is dominated by the dynamics of molecular rearrangementsaccompanying ET at the AZ-SAM interface. Theseinclude internal electric field-dependent low-amplitudeprotein motions and the reorganization of interfacial watermolecules, but not protein reorientation. Interfacialmolecular dynamics also control the kinetics of shortrangeET for electrostatically and covalently immobilizedcytochrome c. This mechanism therefore may be utilizedfor short-distance ET irrespective of the type of metalcenter, the surface electrostatic potential, and the nature ofthe protein−SAM interaction.
2011
- Cloning, Expression and Physico-Chemical Characterization of a New Di-Heme Cytochrome c from Shewanella baltica OS155.
[Articolo su rivista]
DI ROCCO, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Ferrari, Erika; Monari, Stefano; Sola, Marco
abstract
The 16 kDa di-heme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in E. coli and investigated through UV-Vis, MCD and 1H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides di-heme cytochrome c (DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three -helices and a compact overall folding. The C-terminal domain is less helical, as the corresponding domain of R. sphaeroides DHC. The two heme groups are bridged by Tyr26 in correspondence of the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pKa values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and ionization of the heme propionate-7, respectively. Reduction potentials of -0.144 and -0.257 V (vs SHE), were determined for the C- and N-terminal heme group, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and found to reproduce successfully the ionic strength dependence of E°’.
2011
- Dissociation Dynamics of Asymmetric Alkynyl(Aryl)Iodonium Radicals: An ab Initio DRC Approach to Predict the Surface Functionalization Selectivity
[Articolo su rivista]
Fontanesi, Claudio; Bortolotti, Carlo Augusto; Vanossi, Davide; M., Marcaccio
abstract
The dissociation process of neutral open-shell [4-F—(C6H4)—I—CtC—(CH2)4—Cl] and [4-NO2—(C6H4)—I—CtC—(CH2)4—Cl] asymmetric iodonium radicals was studiedtheoretically. Vertical electron affinities and DRC (dynamic reactioncoordinate) results were obtained and compared with experimentalevidence. In particular, the fluorine and nitro substituent groups wereselected because of (i) their opposite electron-withdrawing/electrondonatingeffects and (ii) experimental evidence that the grafting ability,in terms of alkynyl/aryl grafting ratio, increases with decreasingelectron-withdrawing nature of the para-position substituent on the phenyl ring. DRC results show that the dissociation dynamicsof the iodinealkynyl carbon bond, for the nitro-substituted iodonium, occurs on a longer time scale than that of the fluorinesubstitutediodonium. This finding is in agreement with the overall experimental results.
2011
- Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction
[Articolo su rivista]
Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco
abstract
Unfolding turns immobilized cytochrome c into a His–His ligated form endowed with catalytic activity towards O2, which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.
2011
- pH-dependent peroxidase activity of yeast cytochrome c and its triple mutant adsorbed on kaolinite
[Articolo su rivista]
Ranieri, Antonio; Bernini, F.; Bortolotti, Carlo Augusto; Bonifacio, A.; Sergo, V.; Castellini, Elena
abstract
The peroxidase activity of wild type yeast cytochrome c and its triple mutant K72AK73AK79A adsorbed onto kaolinite was investigated as a function of pH and temperature. Both adsorbedproteins displayed an appreciable catalytic activity, which remained constant from pH 7 to 10, decreased below pH 7 and showed a remarkable increase at pH values lower than 4. In the whole pH range investigated the catalytic activity of the adsorbed wild type cytochrome c was higher than that of the mutant. Both diffuse-reflectance UV-Vis and Resonance Raman spectroscopies applied on solid samples were used to probe the structural features responsible for the catalytic activity of the immobilized proteins. At neutral and alkaline pH values a six-coordinate low-spin form of cytochrome c was observed, while at pH < 7 the formation of a high-spin species occurred whose population increased at decreasing pH. The orientation and exposure of the haem to the substrate - strictly dependent on adsorption - was found to affect the peroxidase activity.
2011
- The Reorganization Energy in Cytochrome c is Controlled by the Accessibility of the Heme to the Solvent
[Articolo su rivista]
Bortolotti, Carlo Augusto; Siwko, Magdalena Elzbieta; Castellini, Elena; Ranieri, Antonio; Sola, Marco; S., Corni
abstract
Elucidation of the molecular determinants of the reorganization energy λ is central to the understanding of fundamental biological processes based on energy transduction pathways. Here, we use a combined experimental/theoretical approach to electrochemically determine the reorganization energy for a number of cytochrome c variants and compute structure-related properties relevant to the kinetics of the electron transfer process through molecular dynamics simulations. We find that the exposure of the heme group to solvent controls the reorganization energy of the investigated proteins. Therefore, fine-tuning of the kinetics of the electron transfer process can be achieved through modulation of the accessibility of the iron to the surrounding water. Our findings lead the way for a new strategy for the design of protein-based bioelectronic materials, requiring fast and efficient electron transfer.
2011
- Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study
[Articolo su rivista]
Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Tavagnacco, Claudio; Borsari, Marco
abstract
The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E°’ of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E°’ value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.
2010
- Electron Transfer Properties and Hydrogen Peroxide, Electrocatalysis of Cytochrome c Variants at Positions 67 and 80
[Articolo su rivista]
Casalini, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract
Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome c with a noncoordinatingAla residue and alteration of the hydrogen bonding network in the region nearby following substitution ofTyr67 were investigated as effectors of the thermodynamics and kinetics of the protein-electrode electrontransfer (ET) and the heme-mediated electrocatalytic reduction of H2O2. To this end, the voltammetry of theMet80Ala, Met80Ala/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome c chemisorbedon carboxyalkanethiol self-assembled monolayers was measured at varying temperature and hydrogen peroxideconcentration. The thermodynamic study shows that insertion of His and Ala residues in place of Tyr67results mainly in differences in protein-solvent interactions at the heme crevice with no relevant effects onthe Eo′ values at pH 7, which for single and double variants range from approximately -0.200 to -0.220 V(vs SHE). On the contrary, both double variants show much lower ET rates compared to Met80Ala, mostlikely as a consequence of a change in the ET pathways. In the present nondenaturing immobilizing conditions,and with hydrogen peroxide concentrations in the micromolar range, the variants catalyze H2O2 reduction atthe electrode, whereas wild-type cytochrome c does not. H2O2 electrocatalysis occurs with an efficientmechanism likely involving a fast catalase-like process followed by electrocatalytic reduction of the resultingdioxygen at the electrode. Comparison of Met80Ala/Tyr67His with Met80Ala/Tyr67Ala shows that the presenceof a general acid-base residue for H2O2 recognition and binding through H-bonding in the distal heme siteis a key requisite for the reductive turnover of this substrate.
2010
- Experiments and Models for physics learning in primary school
[Relazione in Atti di Convegno]
Mariani, Cristina; Corni, Federico; Altiero, Tiziana; Bortolotti, Carlo Augusto; Giliberti, Enrico; Landi, Laura; Marchetti, Mauro; A., Martini
abstract
The project “Little scientists in the lab: Experiments & Models for science learning in primary school”, funded by the Ministry of Education and being currently under development in Italy, is addressed to primary school teachers of the Faculty of Science Education and consequently to pupils. This project proposes a “Model-centered Learning Environment” to build pilot activities for teaching and learning science and physics, based on experimental and modeling activities. The approach for teacher training is to assign group tasks that promote learning.We set up a website to support teachers’ school activities to facilitate and promote communication and exchange of materials between teachers and researchers of the University of Modena and Reggio Emilia, as well as between the teachers themselves. In this paper, we illustrate the general features of the project and focus on preliminary results of an in-service training for teachers in fluids and electricity.
2010
- Interaction studies of a β-cysteine functionalized polythiophene with biomolecules
[Abstract in Atti di Convegno]
Bortolotti, Carlo Augusto; F., Filace; Parenti, Francesca; Mucci, Adele; Schenetti, Luisa
abstract
Polythiophenes are an important representative class of conjugated and thermally stable polymers that can be used in alternative to inorganic semiconductors in optical and electronic devices [1]. Recently, they have been studied as biological sensors [2], thanks to the presence of the electroconducting backbone together with β-substituents able to interact with biological species. The interaction between the polymer side chain functionalities and the analyte can be of different type (hydrogen bonds, ionic interactions …) depending on the analyte, or the backbone itself can directly interact with the biomolecule through π-π stacking.In this study, a previously synthesized β-cysteine functionalized polythiophene (PTCys, Fig 1) [3] was investigated as folic acid [4] and cytochrome C (horse heart) biosensor.[1] Skotheim Ed. T., Reynolds J., Elsenbamer R., Dekker Marcel, Inc., New York, NY, USA, Handbook of conducting polymers 2nd Edition, 1998; Skotheim Ed. T., Dekker Marcel, Inc., New York, NY, USA, Handbook of conducting polymers 1986[2] Berggren, M.; Inganas, O.; Gustafsson, G.; Nature,1994, 372, 444-446[3] Cagnoli R., Lanzi M., Mucci A., Parenti F., Schenetti L., Polymer, 2005, 46(11), 3588-3596[4] Zhiyi Yao, Chun Li and Gaoquan Shi, Langmuir,2008, 24, 12829-12835
2010
- Introduzione elementare all’energia: un laboratorio di scienze per insegnanti di scuola primaria
[Relazione in Atti di Convegno]
Altiero, Tiziana; Bortolotti, Carlo Augusto; Corni, Federico; Giliberti, Enrico; R., Greco; Marchetti, Mauro; Mariani, Cristina
abstract
L’insegnamento delle Scienze nella Scuola Primaria deve fornire agli alunni le basi per un approccio scientifico ai fenomeni e ancor prima di proporre temi di discipline separate è opportuno che favorisca la costituzione di un insieme di concetti elementari di fondo comuni e trasversali alle discipline stesse. In ciò gioca un ruolo fondamentale la formazione iniziale degli insegnanti che, anche in considerazione delle linee che si evincono dalle bozze della imminente riforma del Corso di Laurea in Scienze della Formazione Primaria, devono poter usufruire di validi supporti disciplinari, senza però dimenticare la profonda interconnessione delle discipline scientifiche nell’interpretazione dei fenomeni quotidiani e dei processi naturali compresi nel curricolo.Nell’anno accademico 2008-2009 è stato proposto nella Facoltà di Scienze della Formazione un Laboratorio di 16 ore sul tema dell’energia a cui hanno collaborato quattro docenti di diverse discipline scientifiche (Fisica, Chimica, Biologia e Scienze della Terra) e un docente di ambito pedagogico.Il tema dell’energia è stato scelto per il suo carattere di trasversalità alle discipline scientifiche e come contesto paradigmatico in cui costruire un linguaggio e un approccio comune. Il modello energetico adottato si ispira a quello del Karlsruhe Physics Course [1] in cui l’energia è definita a partire dalle sue proprietà di conservazione e introdotta come ente regolatore dei processi naturali. In questo senso viene superata l’idea di “trasformazione dell’energia” introducendo il concetto di “trasferimento di energia” fra portatori, e di esistenza di diverse “forme di energia” pensando piuttosto l’energia come entità sempre associata a un portatore.Il laboratorio è stato aperto con una introduzione sul modello dei portatori e dei trasferitori di energia e si è poi sviluppato con diverse attività di interpretazione da parte degli studenti di situazioni sperimentali riconducibili alle quattro discipline coinvolte. Le attività sperimentali e di interpretazione sono state svolte direttamente dagli studenti, guidati da alcune schede-guida progettare dai singoli docenti e omogenee per tutto il percorso. Prima e dopo l’intervento gli studenti hanno compilato, in modo anonimo, un test di ingresso e un test di uscita.In questo lavoro, dopo una breve introduzione sull’approccio all’energia scelto, verranno presentate in dettaglio la struttura dell’attività del laboratorio e le principali conclusioni che si possono trarre dall’analisi dei materiali prodotti dagli studenti.
2010
- On the Electroreduction Mechanism of Iodonium Salts on Glassy Carbon Electrodes"
[Abstract in Atti di Convegno]
K., Daasbjerg; S. U., Pedersen; Florini, Nicola; Parenti, Francesca; Bortolotti, Carlo Augusto; Vanossi, Davide; Fontanesi, Claudio
abstract
Glassy carbon (GC) surfaces can be functionalized exploiting the electrochemical reduction of iodonium salts of general formula [RIR’]+ [1]. The overall mechanism could be roughly sketched as:
IR’ bond cleavage:[RIR’]+ + e + GC R’GC + IR Route (1)
IR bond cleavage:[RIR’]+ + e + GC RGC + IR’ Route (2)
Upon electroreduction the I-R or the I-R’ bond dissociate, leading to a neutral closed shell organic iodide and an open shell radical, the latter reacts with the GC electrode (grafting). Several factors can influence the R/R’ ratio grafted on the GC surface. In fact, different amounts of the R and R’ radicals can be formed depending on the electronic structure of the neutral open shell [R-I-R’] • radical. Moreover, the different radicals can exhibit different reactivity toward the carbon surface, as well as different electrochemical stabilities (the radical itself could be reduced to a negative closed shell form). To clarify the interplay of the various factors affecting the final surface functionalization, a number of iodoniums has been considered and experimental evidences (electrochemical and XPS) are compared with theoretical results calculated at the DFT level of the theory (electron affinities, potential energy surfaces of competitive reaction pathways).
2010
- On the electroreduction mechanism of iodonium salts on glassy carbon electrodes
[Abstract in Atti di Convegno]
K., Daasbjerg; S. U., Pedersen; DE RENZI, Valentina; Florini, Nicola; Parenti, Francesca; Bortolotti, Carlo Augusto; Vanossi, Davide; Fontanesi, Claudio
abstract
Glassy carbon (GC) surfaces can be functionalized exploiting the electrochemical reduction of iodonium salts of general formula [RIR’]+ [1]. The overall mechanism could be roughly sketched as:
IR’ bond cleavage: [RIR’]+ + e + GC R’GC + IR Route (1)
IR bond cleavage: [RIR’]+ + e + GC RGC + IR’ Route (2)
Upon electroreduction the I-R or the I-R’ bond dissociate, leading to a neutral closed shell organic iodide and an open shell radical, the latter reacts with the GC electrode (grafting). Several factors can influence the R/R’ ratio grafted on the GC surface. In fact, different amounts of the R and R’ radicals can be formed depending on the electronic structure of the neutral open shell [R-I-R’] • radical. Moreover, the different radicals can exhibit different reactivity toward the carbon surface, as well as different electrochemical stabilities (the radical itself could be reduced to a negative closed shell form). To clarify the interplay of the various factors affecting the final surface functionalization, a number of iodoniums has been considered and experimental evidences (electrochemical and XPS) are compared with theoretical results calculated at the DFT level of the theory (electron affinities, potential energy surfaces of competitive reaction pathways).
2010
- Redox properties of heme peroxidases
[Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Bortolotti, Carlo Augusto; Sola, Marco
abstract
Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction 24of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and 25inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, 26involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of 27the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the 28reduction potentials of three redox couples, namely Compound I/Fe3+, Compound I/Compound II and 29Compound II/Fe3+. In particular, the oxidative power of heme peroxidases is controlled by the (high) 30reduction potential of the latter two couples. Moreover, the rapid H2O2-mediated two-electron oxidation 31of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on 32the reduction potential of the Fe3+/Fe2+ couple. The understanding of the molecular determinants of the 33reduction potentials of the above redox couples is crucial for the comprehension of the molecular deter- 34minants of the catalytic properties of heme peroxidases. 35This review provides an overview of the data available on the redox properties of Fe3+/Fe2+, Compound 36I/Fe3+, Compound I/Compound II and Compound II/Fe3+ couples in native and mutated heme peroxidases. 37The influence of the electron donor properties of the axial histidine and of the polarity of the heme envi- 38ronment is analyzed and the correlation between the redox properties of the heme group with the cat- 39alytic activity of this important class of metallo-enzymes is discussed
2009
- Experimental and theoretical investigation of the p- and n-doped states of alkylsulfanyl octithiophenes
[Abstract in Atti di Convegno]
Fontanesi, Claudio; C., Bruno; M., Marcaccio; F., Paolucci; Benassi, Rois; Bortolotti, Carlo Augusto; Mucci, Adele; Parenti, Francesca; Preti, Lisa; Schenetti, Luisa
abstract
Thiophene based materials, oligothiophenes as well, are promising candidates for technological applications, being a possible alternative to high-cost traditional materials for electronics such as silicon. Thus, in this work the charge-transfer properties oftwo structurally related (Si and S2 as sketched on the left) octithiophenes are studied by means of cyclic voltammetryr”(CV). Digital simulations of CV data ,I— , suggest the reversible formation of up to the dication, for both Si and S2, andup to the 4- (Si) and 3- (S2) anions. The ‘ ‘electronic structure of the Si species (2+, i+, neutral, 1-, 2-) is probed by UV/Vis-NIR “in-situ” spectroelectrochemistry. Experimental results are compared to standard potentials calculated at the B3LYP/cc-pVTZ level of the theory and to electronic transition energy and oscillator strength data reckoned by time dependent B3LYP/6 calculations. The consistency observed between experimental and theoretical results indicates the existence, as stable species, of the hypothesized high-spin/high-charge p- and n-doped electronic states.
2009
- Experimental and theoretical investigation of the p- and n-doped states of alkylsulfanyl octithiophenes
[Abstract in Atti di Convegno]
Fontanesi, Claudio; C., Bruno; M., Marcaccio; F., Paolucci; Benassi, Rois; Bortolotti, Carlo Augusto; Mucci, Adele; Parenti, Francesca; Preti, Lisa; Schenetti, Luisa
abstract
We characterizad the charge-transfer properties of two octithiophenes by means of cylcic voltammetry, UV-Vis-NIR spectroelectrochemistry and DFT calculations.
2009
- Phase-Transition-Induced protein redistribution in Lipid Bilayers
[Articolo su rivista]
H. M., Seeger; Bortolotti, Carlo Augusto; Alessandrini, Andrea; P., Facci
abstract
We report an atomic force microscopy study on the lateral spatial redistribution of an integral membraneprotein reconstituted in supported lipid bilayers (SLBs) subjected to a thermally induced phase transition.KcsA proteins were reconstituted in proteoliposomes of POPE/POPG (3:1, mol/mol), and SLBs, includingthe proteins, were then obtained by the vesicle fusion technique on mica. By decreasing the temperature, thelipid bilayer passed from a liquid disordered (ld) phase in which the proteins are homogeneously distributedto a coexistence of solid ordered (so) and ld domains with the proteins preferentially distributed in the lddomains. The inhomogeneous distribution eventually led to protein clustering. The obtained results are discussedin light of the role that the lipid/protein interaction can have in determining the function of integral membraneproteins.
2008
- Electron Transfer and Electrocatalytic Properties of the Immobilized Methionine80Alanine Cytochrome c Variant
[Articolo su rivista]
Casalini, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; Ranieri, Antonio; Sola, Marco
abstract
The M80A variant of yeast iso-1-cytochrome c (cytc), which features a non-coordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM), and investigated by cyclic voltammetry at varying conditions of temperature, pH and O2 concentration. The E°’ value of M80A cytc on both SAMs is of approximately -200 mV (vs. SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous ET are dominated by the presence of an hydroxide ion as sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is the main responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and sets the premises for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.
2008
- Ultraflat Nickel Substrates for Scanning Probe Microscopy of Polyhistidine-Tagged Proteins
[Articolo su rivista]
Alessandrini, Andrea; Bortolotti, Carlo Augusto; G., Bertoni; A., Vezzoli; P., Facci
abstract
A novel approach to the preparation of ultra-flat Nickel substrates suitable to Scanning Probe Microscopy imaging of immobilized polyhistidine-tagged proteins has been devised. Exploiting freshly cleaved mica, Ni thermal evaporation followed by thermal annealing in vacuum, and the template stripping method, we have obtained Ni substrates with a rms roughness as low as 0.12 nm, which bind readily polyhistidine-tagged proteins, enabling molecular resolution imaging of isolated molecules as well as of molecular sub-monolayers. Protein sample exposure to imidazole causes removal of the adsorbates, confirming the involvement of the poly-histidine tail in protein surface immobilization.
2007
- Effects of mutational (Lys to Ala) surface charge changes on the redox properties of electrode-immobilized cytochrome c
[Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract
Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surfacelysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, andthe thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction weredetermined by voltammetry. The reaction thermodynamics were also measured for the same species freelydiffusing in solution. The selected lysine residues surround the heme group and contribute to the positivelycharged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coatedsurfaces. The E°¢ (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for thecorresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM.For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significantchanges in the E°¢ values, which do not simply follow the Coulomb law. The results suggest that the speciesdependentorientation of the protein (and thereby of the heme group) toward the negatively charged SAMinfluences the electrostatic interaction and the resulting E°¢ change. Moreover, these charge suppressionsmoderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donoracceptordistance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.
2007
- Orientation-dependent kinetics of heterogeneous electron transfer for cytochrome c immobilized on gold: Electrochemical determination and theoretical prediction
[Articolo su rivista]
Bortolotti, Carlo Augusto; Borsari, Marco; Sola, Marco; R., Chertkova; D., Dolgikh; A., Kotlyar; P., Facci
abstract
Abstract:A systematic comparison between electron-transfer rate constants measured electrochemically for different cysteine-bearing mutants of cytochrome c chemisorbed on gold surfaces in different orientations has been performed. Experimental data have been correlated with electronic coupling theoretical estimates obtained from two different empirical models for the kinetics of protein electron transfer, the tunneling pathway model and the average packing density model. The results indicate that both models also hold in the case of immobilized redox proteins, outlining their role in the rational design of optimized electron-transfer-based bioinorganic interfaces.
2007
- Redox reactivity of the heme Fe3+/Fe2+ couple in native myoglobins and mutants with peroxidase-like activity
[Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; L., Casella; Bortolotti, Carlo Augusto; E., Monzani; R., Roncone; Sola, Marco
abstract
The reaction enthalpy and entropy for the one-electron reduction of the ferric heme in horse heart and sperm whale aquometmyoglobins (Mb) have been determined exploiting a spectroelectrochemical approach. Also investigated were the T67R, T67K, T67R/S92D and T67R/S92D Mb-H variants (the latter containing a protoheme-l</Emphasis>-histidine methyl ester) of sperm whale Mb, which feature peroxidase-like activity. The reduction potential (E°′) in all species consists of an enthalpic term which disfavors Fe3+ reduction and a larger entropic contribution which instead selectively stabilizes the reduced form. This behavior differs from that of the heme redox enzymes and electron transport proteins investigated so far. The reduction thermodynamics in the series of sperm whale Mb variants show an almost perfect enthalpy-entropy compensation, indicating that the mutation-induced changes in <EquationSource Format="TEX"><![CDATA[$$ Delta H^{{{^circ }ifmmode{'}else$'$fi }}_{{{text{rc}}}} ;{text{and }}Delta S^{{{^circ }ifmmode{'}else$'$fi }}_{{{text{rc}}}} {text{ }} $$]]></EquationSource> are dominated by reduction-induced solvent reorganization effects. The modest changes in E°′ originate from the enthalpic effects of the electrostatic interactions of the heme with the engineered charged residues. The small influence that the mutations exert on the reduction potential of myoglobin suggests that the increased peroxidase activity of the variants is not related to changes in the redox reactivity of the heme iron, but are likely related to a more favored substrate orientation within the distal heme cavity.
2006
- The redox chemistry of the covalently immobilized native and low-pH forms of yeast iso-1-cytochrome c
[Articolo su rivista]
Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Borsari, Marco; P., Facci; Ranieri, Antonio; Sola, Marco
abstract
Cyclic voltammetry experiments were carried out on native Saccharomyces cerevisiae iso-1-cytochrome c and its C102T/N62C variant immobilized on bare polycrystalline gold electrode through the S-Au bond formed by a surface cysteine. Experiments were carried out at different temperatures (5-65 degrees C) and pH values (1.5-7). The E-o' value at pH 7 (+370 mV vs SHE) is approximately 100 mV higher than that for the protein in solution. This difference is enthalpic in origin and is proposed to be the result of the electrostatic repulsion among the densely packed molecules onto the electrode surface. Two additional electrochemical waves are observed upon lowering the pH below 5 (E-o' = +182 mV) and 3 (E-o', = +71 mV), which are attributed to two conformers (referred to as intermediate and acidic, respectively) featuring an altered heme axial ligation. This is the first determination of the reduction potential for low-pH conformers of cytochrome c in the absence of denaturants. Since the native form of cytochrome c can be restored, bringing back the pH to neutrality, the possibility offered by this transition to reversibly modulate the redox potential of cytochrome c is appealing for bioelectronic applications. The immobilized C102T/N62C variant, which differs from the native protein in the orientation of the heme group with respect to the electrode, shows very similar reduction thermodynamics. For both species, the rate constant for electron transfer between the heme and the electrode increases for the acidic conformer, which is also found to act as a biocatalytic interface for dioxygen reduction.
2004
- Characterization of the solution reactivity of a basic heme peroxidase from Cucumis sativus
[Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Leonardi, Alan; Sola, Marco
abstract
A basic heme peroxidase has been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and H NMR spectra from pH 3 to 11. The protein, as isolated, contains a high-spin ferriheme which in the low pH region is sensitive to two acid-base equilibria with apparent pK(a) values of approximately 5 and 3.6, assigned to the distal histidine and to a heme propionate, respectively. At high pH, a new low-spin species develops with an apparent pK(a) of 11, likely due to the binding of an hydroxide ion to the sixth (axial) coordination position of the Fe(III). A number of acid-base equilibria involving heme propionates and residues in the distal cavity also affect the binding of inorganic anions such as cyanide, azide, and fluoride to the ferriheme, as well as the catalytic activity. The reduction potentials of the native protein and of its cyanide derivative, determined through UV-Vis spectroelectrochemistry, result to be -0.320 +/- 0.015 and -0.412 +/- 0.010V, respectively. Overall, the reactivity of this protein parallels those of other plant peroxidases, especially horseradish peroxidase. However, some differences exist in the acid-base equilibria affecting its reactivity and in the reduction potential, likely as a result of small structural differences in the heme distal and proximal cavities.
2004
- Grabbing Yeast iso-1-cytochrome c by Cys102: an effective approach for the assembly of functionally active metalloprotein carpets
[Articolo su rivista]
Gerunda, Mimmo; Bortolotti, Carlo Augusto; Alessandrini, Andrea; Sola, Marco; Battistuzzi, Gianantonio; P., Facci
abstract
We report an approach for immobilizing iso-l-cytochrome c from Saccharomyces cerevisiae on oxygen exposing surfaces derivatized with SH-terminated silanes. The SH moieties from silanes have been brought to react with the partially buried Cys102, forming an intermolecular disulfide bond which anchored covalently cytochrome c to the surface. The presence of a single cysteine residue on the protein surface imparted a well-defined orientation to the molecular edifice. Molecular constructs obtained with native cytochrome c and with a cysteine-depleted mutant (C102T) have been investigated by means of scanning force microscopy under liquid, which was performed to assay the quality of the molecular carpet, showing that the native protein formed a robust monolayer at the surface, whereas only a negligible amount of physisorbed molecules were detected in the case of a mutant. UV-vis absorption spectroscopy was performed to confirm that immobilization takes place via the Cys102 residue. Linear sweep voltammetric measurements showed retention of the redox activity of the covalently immobilized cytochrome c, confirming the viability of the proposed immobilization method for obtaining monolayers of redox active molecules.