In the [PSI +] prion system, the yeast prion protein Sup35 can form structurally distinct amyloid fibrils that lead to distinct transmissible prion states, or strains. However, our understanding of how different Sup35 fibril structures arise and translate to phenotypic variations is limited. Here, using cryo-EM and single-monomer force spectroscopy with optical tweezers, we reveal the structural basis of yeast prion propagation in four wild-type and S17R mutant variants of Sup35 that underlie different [PSI +] strains. Cryo-EM structures show that the four variants form strikingly distinct fibril structures, which exhibit varying stability and chaperone-accessibility. Force spectroscopy suggests the different distinct fibril structures are derived from distinct monomer conformational ensembles. Further, cryo-EM structures indicate that prion strain strength is correlated with enhanced fibril propagation caused by a combination of low fibril stability and a large separation between the Sup35 fibril core and the Ssa1/Sis1 chaperone-binding region. These results provide a structure-based mechanism for the yeast prion strain phenomenon with implications for understanding amyloid propagation in human neurodegenerative diseases.

Starting from the late 1980’s, scanning probe microscopy has progressively diffused in Italy until today. In this paper, we provide a brief account of the main historical events and a current picture of the distribution of the active groups. A survey was prepared by LimeSurvey, made of six sections asking for personal and institutional data, human resources, equipment available, fields of interest, research projects, educational/dissemination activities, and two relevant publications in the last six years. It turns out that the Italian community includes more than seventy groups and two companies. It is widely diffused, although mostly concentrated near large academic and research institutions, often in locations where prominent Italian researchers have operated. This community is active in many scientific fields and can produce research of high international quality. It shows a wide competence, as proven by the list of research works published in journals ranked within the top 20% class. The diffusion of SPM microscopes in industry is still sporadic, possibly due to extensive collaborations between the research institutions and industries themselves. The authors hope that this work might be useful to the community and beyond, and that it might stimulate the formation of a more structured network.

Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrPC) into amyloidal forms denoted as prions or PrPSc. We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation.

Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to β-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTSingle-Molecule Studies of Intrinsically Disordered ProteinsMarco Brucale*†, Benjamin Schuler*‡, and Bruno Samorì*§View Author Information† Institute for the Study of Nanostructured Materials (ISMN), Italian National Council of Research (CNR), Area della Ricerca Roma1, Via Salaria km 29.3 00015 Monterotondo (Rome), Italy‡ Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland§ Department of Pharmacy and Biotechnology, University of Bologna, Via S. Giacomo 11, 40126 Bologna, Italy*E-mail: [email protected]*E-mail: [email protected]*E-mail: [email protected]Cite this: Chem. Rev. 2014, 114, 6, 3281–3317Publication Date (Web):January 17, 2014Publication History Received31 May 2013Published online17 January 2014Published inissue 26 March 2014https://doi.org/10.1021/cr400297gCopyright © 2014 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views4876Altmetric-Citations100LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (13 MB) Get e-AlertsSUBJECTS:Fluorescence,Fluorescence resonance energy transfer,Molecules,Nanopores,Peptides and proteins Get e-Alerts

Protein folding involves a stochastic search through the configurational energy landscape of the protein to find the native structure. Although most proteins have evolved to fold efficiently into a unique native structure, misfolding (the formation of non-native structures) occurs frequently in vivo. Biophysical studies of protein misfolding and early stage aggregation processes are very complex due to the presence of many conformations and different misfolding routes. Single-molecule approaches have proven to be good methods to evaluate the conformational heterogeneity of biological macromolecules, because they can discern amongst different subpopulations, rare or transient states and their energy barriers. The cellular form of the prion protein (PrPC) is a highly conserved membrane-bound protein that is able to misfold into an infectious conformation (PrPSc), which can form aggregates and fibrils with different biochemical and biological properties. Such conformational polymorphisms have been proposed to reflect the conformational heterogeneity of the monomer. Using Atomic Force Microscope (AFM) force spectroscopy, we investigated the conformational equilibria of mouse (Mo) prion protein (PrP) using two different polymeric protein constructs.

Here, we describe the single molecule force spectroscopy (SMFS)-based experimental protocol we have recently used to single out different classes of conformations in a chimeric multimodular protein containing an intrinsically disordered (human Alpha Synuclein) domain. Details are provided regarding cloning, expression and purification of the chimeric polyprotein constructs, optimal surface preparation, SMFS data collection and filtering. Although the specificity of the issue and the ensemble of nonstandard techniques needed to perform the described procedures render this a rather unorthodox protocol, it is relatively straightforward to adapt it to the study of other protein domains.

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.

We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.

Angewandte Chemie International EditionVolume 50, Issue 19 p. 4394-4397 Communication Single-Molecule-Level Evidence for the Osmophobic Effect† Daniel Aioanei, Daniel Aioanei Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Search for more papers by this authorShanshan Lv, Shanshan Lv Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1 (Canada)Search for more papers by this authorDr. Isabella Tessari, Dr. Isabella Tessari Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58/B, 35121 Padova (Italy)Search for more papers by this authorDr. Aldo Rampioni, Dr. Aldo Rampioni Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Search for more papers by this authorProf. Luigi Bubacco, Prof. Luigi Bubacco Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58/B, 35121 Padova (Italy)Search for more papers by this authorProf. Hongbin Li, Prof. Hongbin Li Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1 (Canada)Search for more papers by this authorProf. Bruno Samorì, Prof. Bruno Samorì Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Search for more papers by this authorDr. Marco Brucale, Corresponding Author Dr. Marco Brucale Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy)Search for more papers by this author Daniel Aioanei, Daniel Aioanei Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Search for more papers by this authorShanshan Lv, Shanshan Lv Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1 (Canada)Search for more papers by this authorDr. Isabella Tessari, Dr. Isabella Tessari Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58/B, 35121 Padova (Italy)Search for more papers by this authorDr. Aldo Rampioni, Dr. Aldo Rampioni Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Search for more papers by this authorProf. Luigi Bubacco, Prof. Luigi Bubacco Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58/B, 35121 Padova (Italy)Search for more papers by this authorProf. Hongbin Li, Prof. Hongbin Li Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1 (Canada)Search for more papers by this authorProf. Bruno Samorì, Prof. Bruno Samorì Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Search for more papers by this authorDr. Marco Brucale, Corresponding Author Dr. Marco Brucale Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy) S3 Center of Nanostructures and Biosystems at Surfaces, Istituto di Nanoscienze—CNR (Italy)Dipartimento di Biochimica, Università di Bologna, Via Irnerio 48, 40126 Bologna (Italy)Search for more papers by this author First published: 06 April 2011 https://doi.org/10.1002/anie.201006714Citations: 22 † We are grateful to Prof. G. D. Rose for critical reading of an earlier version of the manuscript and valuable suggestions. We thank Enkelejda Miho for her help with velocity clamp experiments and Dr. Y. Cao for his help with force-clamp and force-ramp experiments. This work was supported by the Ministero dell'Universitá e della Ricerca—Fondo per gli Investimenti della Ricerca di Base (MIUR—FIRB) RBNE03PX83/001; MIUR—FIRB Progetto NG-lab (G.U. 29/07/05 n.175); Progetti di Ricerca di Interesse Nazionale (PRIN) 2008 (Prot. 2008KZ3E5, Prot. 2008SYP79), by the Natural Sciences and Engineering Research Council of Canada and Canada Research Chairs Program, and by Progetto di Ateneo 2008, Università di Padova. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract Chemical chaperones: Protecting osmolytes play a crucial role in preventing protein denaturation in harsh environmental conditions of living organisms. Experimental evidence is provided for a mechanism of protein-fold stabilization by these molecules that is in accord with the hypothesis of a backbone-based osmophobic effect. (In picture: ΔG=free energy, [O]=osmolyte concentration, χ=unfolding reaction coordinate.) 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Currently, nanotechnology is exposing the properties of DNA in unprecedented detail leading to new insights on the biological behavior and function of DNA. With the structural perfection of a self-ass

Chemical chaperones: Protecting osmolytes play a crucial role in preventing protein denaturation in harsh environmental conditions of living organisms. Experimental evidence is provided for a mechanism of protein‐fold stabilization by these molecules that is in accord with the hypothesis of a backbone‐based osmophobic effect. (In picture: ΔG=free energy, [O]=osmolyte concentration, χ=unfolding reaction coordinate.)

Protecting osmolytes are widespread small organic molecules able to stabilize the folded state of most proteins against various denaturing stresses in vivo. The osmophobic model explains thermodynamically their action through a preferential exclusion of the osmolyte molecules from the protein surface, thus favoring the formation of intrapeptide hydrogen bonds. Few works addressed the influence of protecting osmolytes on the protein unfolding transition state and kinetics. Among those, previous single molecule force spectroscopy experiments evidenced a complexation of the protecting osmolyte molecules at the unfolding transition state of the protein, in apparent contradiction with the osmophobic nature of the protein backbone. We present single-molecule evidence that glycerol, which is a ubiquitous protecting osmolyte, stabilizes a globular protein against mechanical unfolding without binding into its unfolding transition state structure. We show experimentally that glycerol does not change the position of the unfolding transition state as projected onto the mechanical reaction coordinate. Moreover, we compute theoretically the projection of the unfolding transition state onto two other common reaction coordinates, that is, the number of native peptide bonds and the weighted number of native contacts. To that end, we augment an analytic Ising-like protein model with support for group-transfer free energies. Using this model, we find again that the position of the unfolding transition state does not change in the presence of glycerol, giving further support to the conclusions based on the single-molecule experiments.

This chapter contains sections titled: Abstract Introduction Basic Elements of the SMFS Methodology for Protein Folding Studies the SMFS Methodology in the Studies of the Mechanical Properties and Interactions of IDPs Probing the Conformational Equilibria of the IDPs Involved in Neurodegenerative Diseases: the Case of α-Synuclein Future Perspectives References

Label-free DNA detection plays a crucial role in developing point-of-care biochips. Capacitance detection is a promising technology for label-free detection. However, data published in literature often show evident time drift, large standard deviation, scattered data points, and poor reproducibility. To address these problems, mercapto-hexanol or similar alkanethiols are usually considered as blocking agents. The aim of the present paper is to investigate new blocking agents to further improve DNA probe surfaces. Data from AFM, SPR, florescence microscopy, and capacitance measurements are used to investigate new lipoate and ethylene-glycol molecules. The new surfaces offer further improvements in terms of diminished detection errors. Film structures are investigated at the nano-scale to justify the detection improvements in terms of probe surface quality. This study demonstrates the superiority of lipoate and ethylene-glycol molecules as blocking candidates when immobilizing molecular probes onto spot surfaces in label-free DNA biochip.