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Dipartimento di Scienze Fisiche, Informatiche e Matematiche sede ex-Fisica

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2023 - Applying optical tweezers technology to reveal Thymidylate Synthase interactions with its consensus mRNA [Abstract in Atti di Convegno]
Malpezzi, Giulia; Viader, Xvaier; Zaltron, Annamaria; Neri, Benedetta; di Felice, Giulia; Tagliazucchi, Lorenzo; Aiello, Daniele; Moschella, MARIA GAETANA; Costi, Maria Paola; D'Arca, Domenico; Cecconi, Ciro

Malpezzi, Giulia; Viander, Xavier; Zaltron, Annamaria; Neri, Benedetta; Di Felice, Giulia; Tagliazucchi, Lorenzo; Aiello, Daniele; Moschella, Maria; Costi, Maria Paola; D’Arcae, Domenico; Cecconi, Ciro

2023 - Exploiting the Optical Tweezer technology to reveal thymidylate synthase interactions with its consensus mRNA [Abstract in Atti di Convegno]
Malpezzi, Giulia; Viander, Xavier; Zaltron, Annamaria; Neri, Benedetta; Di Felice, Giulia; Tagliazucchi, Lorenzo; Aiello, Daniele; Moschella, MARIA GAETANA; Costi, Maria Paola; D'Arca, Domenico; Cecconi, Ciro

2023 - Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process [Articolo su rivista]
Choudhary, Dhawal; Mediani, Laura; Avellaneda, Mario J; Bjarnason, Sveinn; Alberti, Simon; Boczek, Edgar E; Heidarsson, Pétur O; Mossa, Alessandro; Carra, Serena; Tans, Sander J; Cecconi, Ciro

: Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

2023 - The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins [Articolo su rivista]
Ecroyd, Heath; Bartelt-Kirbach, Britta; Ben-Zvi, Anat; Bonavita, Raffaella; Bushman, Yevheniia; Casarotto, Elena; Cecconi, Ciro; Lau, Wilson Chun Yu; Hibshman, Jonathan D; Joosten, Joep; Kimonis, Virginia; Klevit, Rachel; Liberek, Krzysztof; Mcmenimen, Kathryn A; Miwa, Tsukumi; Mogk, Axel; Montepietra, Daniele; Peters, Carsten; Rocchetti, Maria Teresa; Saman, Dominik; Sisto, Angela; Secco, Valentina; Strauch, Annika; Taguchi, Hideki; Tanguay, Morgan; Tedesco, Barbara; Toth, Melinda E; Wang, Zihao; Benesch, Justin L P; Carra, Serena

: The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

2022 - A fit-less approach to the elasticity of the handles in optical tweezers experiments [Articolo su rivista]
Mossa, Alessandro; Cecconi, Ciro

The elastic properties of the double-stranded DNA handles used in optical tweezers experiments on biomolecules are customarily modeled by an extensible worm-like chain model. Fitting such a model to experimental data, however, is no trivial task, as the function depends on four parameters in a highly non-linear fashion. We hereby propose a method to bypass the fitting procedure and obtain an empirical force vs. extension curve that accurately reproduces the elasticity of the handles.

2022 - Combining enhanced sampling and deep learning dimensionality reduction for the study of the heat shock protein B8 and its pathological mutant K141E [Articolo su rivista]
Montepietra, Daniele; Cecconi, Ciro; Brancolini, Giorgia

The biological functions of proteins closely depend on their conformational dynamics. This aspect is especially relevant for intrinsically disordered proteins (IDP) for which structural ensembles often offer more useful representations than individual conformations. Here we employ extensive enhanced sampling temperature replica-exchange atomistic simulations (TREMD) and deep learning dimensionality reduction to study the conformational ensembles of the human heat shock protein B8 and its pathological mutant K141E, for which no experimental 3D structures are available. First, we combined homology modelling with TREMD to generate high-dimensional data sets of 3D structures. Then, we employed a recently developed machine learning based post-processing algorithm, EncoderMap, to project the large conformational data sets into meaningful two-dimensional maps that helped us interpret the data and extract the most significant conformations adopted by both proteins during TREMD. These studies provide the first 3D structural characterization of HSPB8 and reveal the effects of the pathogenic K141E mutation on its conformational ensembles. In particular, this missense mutation appears to increase the compactness of the protein and its structural variability, at the same time rearranging the hydrophobic patches exposed on the protein surface. These results offer the possibility of rationalizing the pathogenic effects of the K141E mutation in terms of conformational changes.

2021 - Designing selective Cys-ligands to unpair the binding of the Human Transcription Enhancer Associated Domain 4 (hTEAD-4) with its modulators to halt cancer cell growth [Poster]
Tagliazucchi, Lorenzo; Malpezzi, Giulia; Pozzi, Cecilia; Lopresti, Ludovica; Venturelli, Alberto; D'Arca, Domenico; Marverti, Gaetano; Cecconi, Ciro; Ponterini, Glauco; Costi, Maria Paola

The Hippo Signalling cascade is an emerging target in tumour suppression regulation, neoplastic hypertrophy, and regenerative medicine. The pathway is activated by circulating anti-proliferative signals which leads to the phosphorylation of Yes Associated Protein (hYAP1) on Ser127/381, thus 14-3-3σ mediated cytosolic retention. Genetic alterations or exogenous factor may cause YAP nuclear migration and association to TEAD1-4 (Transcription Enhancer Associated Domain), triggering up-regulation of anti-apoptotic genes [1]. hTEAD is an enhancer that activates the nuclear transcription of genes as EMT’s, EGFR and cyclins, and promotes the synthesis of survivin, tyrosine kinase HER3, and mitochondrial Bcl-xL involved in cell proliferation. TEAD binds a palmitic (palm) or myristic (myr) acids, tethered at Cys367 pocket, however its biological role is still not well known. hTEAD isoform-4 is the most represented of its family in solid tumours and its overexpression or mutation leads to cancer development and metastasis. Recent studies have considered hTEAD a promising target for anticancer drugs. Its inhibition strategy includes the disruption/prevention of YAP1:TEAD4 complex formation [2]. With the aim to develop a specific cysteine-directed inhibition strategy, we studied Cys on the protein surface and investigated their reactivity. Hence, our studies focus on characterizing the recombinant hTEAD4-ybd (aa217-434) surface though the analysis of the reactivity of its four Cys thiols (Cys310, Cys335, Cys367, Cys410), all close to YAP binding area. First, myr-Cys-367 was investigated to confirm the auto-myristoilation of the E. coli recombinant hTEAD4 through RP-chromatography on UHPLC-Orbitrap Q-Ex (ThermoFisher™) by multicharged TIC deconvolution, and the total myr-TEAD was assessed around 25%. Myristate position was confirmed by FASP protein tryptic hydrolysis and tandem-MS peptide analysis. We studied hTEAD binding of a small disulphides and thiols library with different chemical properties through the exposed cysteines residues in presence of different concentration of reducing agent [3]. Top8 DDA (HCD)-MS/MS scan on the tryptic peptides suggested the ligands’ high selectivity towards Cys335. Cys367 was never found conjugated, even in the non-Myr fraction, hinting the low accessibility to the lipid pocket. The number of surface reactive Cys was confirmed by a reverse-titration of the protein against increasing amount of thiophenol; excess of unreacted thiophenol was measured by HPLC-UV-ELSD (Agilent™ 1260), suggesting a 1:1 stoichiometry. We confirmed hTEAD-ybd ligand ratio by fluoresceine labelling with absorption and fluorescence differential spectroscopy. The ongoing work engages the screening of a larger compound library to study YAP:TEAD interaction with a ligand displacement assay of labelled TEAD to a rhodamine-tagged peptidomimetic probe to achieve structural information of the heterodimer interface and to start a hit-optimization programme. REFERENCES [1] Santucci M, Vignudelli T, et al. The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment. J Med Chem. 2015 Jun 25;58(12):4857-73. [2] Elisi G.M, Santucci M, et al. Repurposing of Drugs Targeting YAP-TEAD Functions. Cancers 2018, 10, 329. [3] Malpezzi G MSc Degree Thesis, Solvent exposure, and reactivity of the cysteines of Transcription Enhancer Associate Domain (TEAD), a potential anticancer target, 2021. University of Pavia – University of Modena and Reggio Emilia.

2021 - From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers [Articolo su rivista]
Heidarsson, Pétur O; Cecconi, Ciro

Single-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand-receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics - from protein synthesis to physiological function - as well as emerging future issues that are beginning to be addressed with novel approaches.

2020 - Effects of Ligand Binding on the Energy Landscape of Acyl-CoA-Binding Protein [Articolo su rivista]
Sonar, P.; Bellucci, L.; Mossa, A.; Heidarsson, P. O.; Kragelund, B. B.; Cecconi, C.

Binding of ligands is often crucial for function yet the effects of ligand binding on the mechanical stability and energy landscape of proteins are incompletely understood. Here, we use a combination of single-molecule optical tweezers and MD simulations to investigate the effect of ligand binding on the energy landscape of acyl-coenzyme A (CoA)-binding protein (ACBP). ACBP is a topologically simple and highly conserved four-α-helix bundle protein that acts as an intracellular transporter and buffer for fatty-acyl-CoA and is active in membrane assembly. We have previously described the behavior of ACBP under tension, revealing a highly extended transition state (TS) located almost halfway between the unfolded and native states. Here, we performed force-ramp and force-jump experiments, in combination with advanced statistical analysis, to show that octanoyl-CoA binding increases the activation free energy for the unfolding reaction of ACBP without affecting the position of the transition state along the reaction coordinate. It follows that ligand binding enhances the mechanical resistance and thermodynamic stability of the protein, without changing its mechanical compliance. Steered molecular dynamics simulations allowed us to rationalize the results in terms of key interactions that octanoyl-CoA establishes with the four α-helices of ACBP and showed that the unfolding pathway is marginally affected by the ligand. The results show that ligand-induced mechanical stabilization effects can be complex and may prove useful for the rational design of stabilizing ligands.

2020 - Studying heat shock proteins through single-molecule mechanical manipulation [Articolo su rivista]
Choudhary, D.; Mediani, L.; Carra, S.; Cecconi, C.

Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as “sponges” that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at the single-molecule level the structural dynamics of HSPs and sHSPs and to examine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed.

2019 - Bio-molecular applications of recent developments in optical tweezers [Articolo su rivista]
Choudhary, Dhawal; Mossa, Alessandro; Jadhav, MILIND SURESH; Cecconi, Ciro

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

2019 - Small heat shock proteins: multifaceted proteins with important implications for life [Articolo su rivista]
Carra, Serena; Alberti, Simon; Benesch, Justin L. P.; Boelens, Wilbert; Buchner, Johannes; Carver, John A.; Cecconi, Ciro; Ecroyd, Heath; Gusev, Nikolai; Hightower, Lawrence E.; Klevit, Rachel E.; Lee, Hyun O.; Liberek, Krzysztof; Lockwood, Brent; Poletti, Angelo; Timmerman, Vincent; Toth, Melinda E.; Vierling, Elizabeth; Wu, Tangchun; Tanguay, Robert M.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world’s experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26–29 August 2018.

2018 - The complex conformational dynamics of neuronal calcium sensor-1: A single molecule perspective [Articolo su rivista]
Choudhary, Dhawal; Kragelund, Birthe B.; Heidarsson, Pétur O.; Cecconi, Ciro

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca 2+ changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca 2+ channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca 2+ -bound, Mg 2+ -bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca 2+ and Mg 2+ ) to its EF-hands. At pathologically high Ca 2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties.

2016 - Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level [Articolo su rivista]
Alemany, Anna; Rey Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.

2015 - A novel mechano-enzymatic cleavage mechanism underlies transthyretin amyloidogenesis [Articolo su rivista]
Marcoux, Julien; Mangione, P. Patrizia; Porcari, Riccardo; Degiacomi, Matteo T; Verona, Guglielmo; Taylor, Graham W; Giorgetti, Sofia; Raimondi, Sara; Sanglier Cianférani, Sarah; Benesch, Justin LP; Cecconi, Ciro; Naqvi, Mubarak Mohsin; Gillmore, Julian D; Hawkins, Philip N; Stoppini, Monica; Robinson, Carol V; Pepys, Mark B; Bellotti, Vittorio

The mechanisms underlying transthyretin-related amyloidosis in vivo remain unclear. The abundance of the 49-127 transthyretin fragment in ex vivo deposits suggests that a proteolytic cleavage has a crucial role in destabilizing the tetramer and releasing the highly amyloidogenic 49-127 truncated protomer. Here, we investigate the mechanism of cleavage and release of the 49-127 fragment from the prototypic S52P variant, and we show that the proteolysis/fibrillogenesis pathway is common to several amyloidogenic variants of transthyretin and requires the action of biomechanical forces provided by the shear stress of physiological fluid flow. Crucially, the non-amyloidogenic and protective T119M variant is neither cleaved nor generates fibrils under these conditions. We propose that a mechano-enzymatic mechanism mediates transthyretin amyloid fibrillogenesis in vivo. This may be particularly important in the heart where shear stress is greatest; indeed, the 49-127 transthyretin fragment is particularly abundant in cardiac amyloid. Finally, we show that existing transthyretin stabilizers, including tafamidis, inhibit proteolysis-mediated transthyretin fibrillogenesis with different efficiency in different variants; however, inhibition is complete only when both binding sites are occupied.

2015 - Single-Molecule Folding Mechanisms of the apo- and Mg(2+)-Bound States of Human Neuronal Calcium Sensor-1 [Articolo su rivista]
Naqvi, Mohsin M; Heidarsson, Petur O; Otazo, Mariela R; Mossa, Alessandro; Kragelund, Birthe B; Cecconi, Ciro

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca(2+) concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca(2+)-activated state of NCS proteins, little is known about the conformational dynamics of the Mg(2+)-bound and apo states, both of which are populated, at least transiently, at resting Ca(2+) conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg(2+) and in the absence of divalent ions. Under tension, the Mg(2+)-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca(2+)-bound state, the presence of Mg(2+) allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.

2014 - Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1 [Articolo su rivista]
Heidarsson, Petur O.; Naqvi, Mohsin M.; Otazo, Mariela R.; Mossa, Alessandro; Kragelund, Birthe B.; Cecconi, Ciro

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.

2014 - The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations [Articolo su rivista]
Caldarini, M.; Sonar, PUNAM SURESH; Samidass, VALPAPURAM IMMANUEL; Tavella, D.; Volonte, C.; Pandini, V.; Vanoni, M. A.; Aliverti, A.; Broglia, R. A.; Tiana, G.; Cecconi, Ciro

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long-lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Refolding of mechanically denatured HIV-1-PR monomers is also a multiple-pathway process. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, and provide information regarding possible structural features of the PF state. (C) 2014 Elsevier B.V. All rights reserved.

2013 - Conformational Dynamics of Single Protein Molecules Studied by Direct Mechanical Manipulation [Capitolo/Saggio]
Heidarsson, Petur O.; Naqvi, Mohsin M.; Sonar, PUNAM SURESH; Samidass, VALPAPURAM IMMANUEL; Cecconi, Ciro

Advances in single-molecule manipulation techniques have recently enabled researchers to study a growing array of biological processes in unprecedented detail. Individual molecules can now be manipulated with subnanometer precision along a simple and well-defined reaction coordinate, the molecular end-to-end distance, and their conformational changes can be monitored in real time with ever-improving time resolution. The behavior of biomolecules under tension continues to unravel at an accelerated pace and often in combination with computational studies that reveal the atomistic details of the process under investigation. In this chapter, we explain the basic principles of force spectroscopy techniques, with a focus on optical tweezers, and describe some of the theoretical models used to analyze and interpret single-molecule manipulation data. We then highlight some recent and exciting results that have emerged from this research field on protein folding and protein-ligand interactions.

2013 - Single-Molecule Folding Mechanism of an EF-Hand Neuronal Calcium Sensor [Articolo su rivista]
Heidarsson, Petur O.; Otazo, Mariela R.; Bellucci, Luca; Mossa, Alessandro; Imparato, Alberto; Paci, Emanuele; Corni, Stefano; DI FELICE, Rosa; Kragelund, Birthe B.; Cecconi, Ciro

EF-hand calcium sensors respond structurally to changes in intracellular Ca2+ concentration, triggering diverse cellular responses and resulting in broad interactomes. Despite impressive advances in decoding their structure-function relationships, the folding mechanism of neuronal calcium sensors is still elusive. We used single-molecule optical tweezers to study the folding mechanism of the human neuronal calcium sensor 1 (NCS1). Two intermediate structures induced by Ca2+ binding to the EF-hands were observed during refolding. The complete folding of the C domain is obligatory for the folding of the N domain, showing striking interdomain dependence. Molecular dynamics results reveal the atomistic details of the unfolding process and rationalize the different domain stabilities during mechanical unfolding. Through constant-force experiments and hidden Markov model analysis, the free energy landscape of the protein was reconstructed. Our results emphasize that NCS1 has evolved a remarkable complex interdomain cooperativity and a fundamentally different folding mechanism compared to structurally related proteins.

2013 - Structure, Folding Dynamics, and Amyloidogenesis of D76N beta(2)-Microglobulin ROLES OF SHEAR FLOW, HYDROPHOBIC SURFACES, AND alpha-CRYSTALLIN [Articolo su rivista]
Mangione, P. Patrizia; Esposito, Gennaro; Relini, Annalisa; Raimondi, Sara; Porcari, Riccardo; Giorgetti, Sofia; Corazza, Alessandra; Fogolari, Federico; Penco, Amanda; Goto, Yuji; Lee, Young Ho; Yagi, Hisashi; Cecconi, Ciro; Naqvi, Mohsin M.; Gillmore, Julian D.; Hawkins, Philip N.; Chiti, Fabrizio; Rolandi, Ranieri; Taylor, Graham W.; Pepys, Mark B.; Stoppini, Monica; Bellotti, Vittorio

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human beta(2)-microglobulin (beta(2)m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N beta(2)m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type beta(2)m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.

2012 - A Highly Compliant Protein Native State with a Spontaneous-like Mechanical Unfolding Pathway [Articolo su rivista]
Heidarsson, Petur O.; Samidass, VALPAPURAM IMMANUEL; Camilloni, Carlo; Imparato, Alberto; Tiana, Guido; Poulsen, Flemming M.; Kragelund, Birthe B.; Cecconi, Ciro

The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force of the four-a-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal pulling axes, with transition states located almost halfway between the unfolded and folded states. Surprisingly, the deformability of ACBP is greater than that observed for the highly pliant molten globule intermediate states. Furthermore, when manipulated from the N- and C-termini, ACBP unfolds by populating a transition state that resembles that observed during chemical denaturation, both for structure and position along the reaction coordinate. Our data provide the first experimental evidence of a spontaneous-like mechanical unfolding pathway of a protein. The mechanical behavior of ACBP is discussed in terms of topology and helix propensity.

2012 - Exploring Folding Pathways of Single Proteins Using Mechanical Manipulation [Relazione in Atti di Convegno]
Heidarsson, Petur O.; Cecconi, Ciro

Protein folding is still a major area of active research. Despite significant progress in understanding the underlying principles, we still cannot efficiently predict the folding mechanism for even a moderately sized protein. Proteins are generally thought to fold by diffusion over a three-dimensional energy landscape. Traditional bulk methods have proven to be very powerful in the study of the folding process but they often suffer from inherent ensemble averaging. Single molecule techniques open up new vistas for studying protein folding, allowing direct analysis of the distribution of events that characterize the heterogeneous folding process. Recently it has become possible to directly manipulate individual proteins using optical tweezers. Here we illustrate the experimental strategy and how this approach has provided a fresh perspective on the protein folding problem.

2011 - DNA molecular handles for single-molecule protein-folding studies by optical tweezers [Capitolo/Saggio]
Cecconi, Ciro; Shank, Elizabeth A; Marqusee, Susan; Bustamante, Carlos

In this chapter, we describe a method that extends the use of optical tweezers to the study of the folding mechanism of single protein molecules. This method entails the use of DNA molecules as molecular handles to manipulate individual proteins between two polystyrene beads. The DNA molecules function as spacers between the protein and the beads, and keep the interactions between the tethering surfaces to a minimum. The handles can have different lengths, be attached to any pair of exposed cysteine residues, and be used to manipulate both monomeric and polymeric proteins. By changing the position of the cysteine residues on the protein surface, it is possible to apply the force to different portions of the protein and along different molecular axes. Circular dichroism and enzymatic activity studies have revealed that for many proteins, the handles do not significantly affect the folding behavior and the structure of the tethered protein. This method makes it possible to study protein folding in the physiologically relevant low-force regime of optical tweezers and enables us to monitor processes - such as refolding events and fluctuations between different molecular conformations - that could not be detected in previous force spectroscopy experiments.

2010 - The folding cooperativity of a protein is controlled by its chain topology [Articolo su rivista]
Shank, Elizabeth A.; Cecconi, Ciro; Dill, Jesse W.; Marqusee, Susan; Bustamante, Carlos

The three-dimensional structures of proteins often show a modular architecture comprised of discrete structural regions or domains. Cooperative communication between these regions is important for catalysis, regulation and efficient folding; lack of coupling has been implicated in the formation of fibrils and other misfolding pathologies(1). How different structural regions of a protein communicate and contribute to a protein's overall energetics and folding, however, is still poorly understood. Here we use a single-molecule optical tweezers approach to induce the selective unfolding of particular regions of T4 lysozyme and monitor the effect on other regions not directly acted on by force. We investigate how the topological organization of a protein (the order of structural elements along the sequence) affects the coupling and folding cooperativity between its domains. To probe the status of the regions not directly subjected to force, we determine the free energy changes during mechanical unfolding using Crooks' fluctuation theorem. We pull on topological variants (circular permutants) and find that the topological organization of the polypeptide chain critically determines the folding cooperativity between domains and thus what parts of the folding/unfolding landscape are explored. We speculate that proteins may have evolved to select certain topologies that increase coupling between regions to avoid areas of the landscape that lead to kinetic trapping and misfolding.

2009 - What can we learn from mechanical unfolding of a single protein domain by optical tweezers [Abstract in Atti di Convegno]
Zhang, C. -Z.; Seog, J.; Dill, J.; Smith, S. B.; Zhang, F. X.; Cecconi, C.; Marqusee, S.; Bustamante, C.; Springer, T. A.

2008 - Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers [Articolo su rivista]
Cecconi, Ciro; Shank, Elizabeth A.; Dahlquist, Frederick W.; Marqusee, Susan; Bustamante, Carlos

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.

2007 - Analysis of P element transposase protein-DNA interactions during the early stages of transposition [Articolo su rivista]
Tang, Mei; Cecconi, Ciro; Bustamante, Carlos; Rio, Donald C.

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.

2007 - Studying protein folding with laser tweezers [Relazione in Atti di Convegno]
Cecconi, C.; Shank, E. A.; Marqusee, S.; Bustamante, C. J.

2005 - Direct observation of the three-state folding of a single protein molecule [Articolo su rivista]
Cecconi, Ciro; Shank, Ea; Bustamante, C; Marqusee, S.

We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia-coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H.

2005 - Guanosine triphosphate acts as a cofactor to promote assembly of initial P-element transposase-DNA synaptic complexes [Articolo su rivista]
Tang, M; Cecconi, Ciro; Kim, H; Bustamante, C; Rio, Dc

P transposable elements in Drosophila are members of a larger class of mobile elements that move using a cut-and-paste mechanism. P-element transposase uses guanosine triphosphate (GTP) as a cofactor for transposition. Here, we use atomic force microscopy (AFM) to visualize protein-DNA complexes formed during the initial stages of P-element transposition. These studies reveal that GTP acts to promote assembly of the first detectable noncovalent precleavage synaptic complex. This initial complex then randomly and independently cleaves each P-element end. These data show that GTP acts to promote protein-DNA assembly, and may explain why P-element excision often leads to unidirectional deletions.

2005 - Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism [Articolo su rivista]
Brown, Ta; Cecconi, Ciro; Tkachuk, An; Bustamante, C; Clayton, Da

The established strand-displacement model for mammalian mitochondrial DNA (mtDNA) replication has recently been questioned in light of new data using two-dimensional (2D) agarose gel electrophoresis. It has been proposed that a synchronous, strand-coupled mode of replication occurs in tissues, thereby casting doubt on the general validity of the "orthodox," or strand-displacement model. We have examined mtDNA replicative intermediates from mouse liver using atomic force microscopy and 2D agarose gel electrophoresis in order to resolve this issue. The data provide evidence for only the orthodox, strand-displacement mode of replication and reveal the presence of additional, alternative origins of lagging light-strand mtDNA synthesis. The conditions used for 2D agarose gel analysis are favorable for branch migration of asymmetrically replicating nascent strands. These data reconcile the original displacement mode of replication with the data obtained from 2D gel analyses.

2001 - Mechanical unfolding of individual T4 lysozyme molecules [Abstract in Rivista]
Yang, Gl; Cecconi, Ciro; Baase, Wa; Matthews, Bw; Dahlquist, R; Bustamante, C.

Mechanical unfolding of individual T4 lysozyme molecules

2000 - Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme [Articolo su rivista]
Yang, Gl; Cecconi, Ciro; Baase, Wa; Vetter, Ir; Breyer, Wa; Haack, Ja; Matthews, Bw; Dahlquist, Fw; Bustamante, C.

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

1998 - Force induced unfolding and refolding of individual protein molecules by SFM [Abstract in Rivista]
Yang, G; Cecconi, Ciro; Haack, J; Bryer, W; Baase, W; Dahlquist, Fw; Kellermayer, Ms; Bustamante, C.

Force induced unfolding and refolding of individual protein molecules by SFM

1997 - Fabrication of hybrid superconductor-semiconductor nanostructures by integrated ultraviolet atomic force microscope lithography [Articolo su rivista]
Pingue, P; Lazzarino, M; Beltram, F; Cecconi, Ciro; Baschieri, P; Frediani, C; Ascoli, C.

Hybrid superconductor-semiconductor (S-Sm) nanostructures were fabricated by integrating standard ultraviolet photolithography and direct patterning of photoresist with an atomic force microscope (AFM). This novel technology was used to fabricate Nb-InAs-Nb weak links comparable in length to the coherence length. These structures exhibit high critical currents up to 10 mu A/mu m in planar geometry at 0.3 K. The fabrication protocol is based on the modification of photolithographically defined patterns by AFM static ploughing of the photoresist. Wet chemical etching is subsequently used for the definition of nanoscale S-Sm-S bridges. Additionally Lift-off procedures allowed the fabrication of submicron superconducting bridges. Successful fabrication of the nanostructures was verified by electrical characterization and by AFM and scanning electron microscope structural characterization. (C) 1997 American Vacuum Society.

1996 - A simple method for preparing calibration standards for the three working axes of scanning probe microscope piezo scanners [Articolo su rivista]
Alliata, D; Cecconi, Ciro; Nicolini, C.

A method for preparing samples suitable for calibrating scanning probe microscopes (SPM) and for eliminating any distortions in images is described. Samples consist of polystyrene particles organized in monolayers and bilayers with hexagonal-ordered domains. The monolayer is not uniform, but is characterized by areas without particles. These discontinuities allow the measurement of the thickness of the monolayer in order to calibrate the z axes, while the lattice constant of the domains can be used as a calibration standard for the x and y axes. The nondeformability of the particles after the deposition on the substrate has been studied by an optical microscope, equipped for interferometric measurements, scanning force microscopy, and scanning tunneling microscopy. The use of these standards directly as substrates for samples is proposed to correct the distortions in the SPM images. (C) 1996 American Institute of Physics.

1996 - Step-up photophobic responses of the unicellular alga Haematococcus pluvialis and their interpretation in terms of photoreceptive apparatus characteristics [Articolo su rivista]
Cecconi, Ciro; Ascoli, C; Petracchi, D.

This paper reports a systematic study of the step-up photophobic responses exhibited by the unicellular alga Haematococcus pluvialis when stimulated unidirectionally or bidirectionally. The stimulus-response curves, obtained at four different wavelengths, are interpreted in terms of the structure of the photoreceptive apparatus. In addition, an indirect method to obtain information about the stigma and photoreceptor(s) is reported. This method is based on the interpolation of experimental with simulated data. Our results confirm the widely accepted view that the photoreceptors are located in the stigma region, and suggest the presence of two photoreceptors.

1995 - Componenti diffrattivi per ottica integrata [Articolo su rivista]
Vergani, A.; Righini, G. C.; Palchetti, L.; Sottini, S.; Cecconi, Ciro

non disponibile

Petracchi, D; Barbi, M; Cecconi, Ciro; Pellegrini, M; Pellegrino, M; Simoni, A.


Simoni, A; Pellegrini, M; Cecconi, Ciro; Pellegrino, M.

Axotomy affects density but not properties of potassium leak channels, in the leech AP neurons