scholarly journals Single-molecule spectroscopy of unfolded proteins and chaperonin action

2014 ◽  
Vol 395 (7-8) ◽  
pp. 689-698 ◽  
Author(s):  
Hagen Hofmann
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Unfolded Proteins ◽  
Conformational Ensembles ◽  
Intrinsically Disordered ◽  
Cellular Environment ◽  
Quantitative Understanding ◽  
Polypeptide Chains

Abstract In the past decade, single-molecule fluorescence techniques provided important insights into the structure and dynamics of proteins. In particular, our understanding of the heterogeneous conformational ensembles of unfolded and intrinsically disordered proteins (IDPs) improved substantially by a combination of FRET-based single-molecule techniques with concepts from polymer physics. A complete knowledge of the forces that act in unfolded polypeptide chains will not only be important to understand the initial steps of protein folding reactions, but it will also be crucial to rationalize the coupling between ligand-binding and folding of IDPs, and the interaction of denatured proteins with molecular chaperones in the crowded cellular environment. Here, I give a personalized review of some of the key findings from my own research that contributed to a more quantitative understanding of unfolded proteins and their interactions with molecular chaperones.

scholarly journals Nanopores to Interrogate the Conformational Ensembles of Intrinsically Disordered Proteins on a Single-Molecule Level

2020 ◽  
Vol 118 (3) ◽  
pp. 214a
Author(s):  
Saurabh Awasthi ◽  
Jared Houghtaling ◽  
Cuifeng Ying ◽  
Aziz Fennouri ◽  
Ivan Shorubalko ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Single Molecule Level ◽  
Conformational Ensembles ◽  
Intrinsically Disordered

scholarly journals Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations

2017 ◽  
Vol 114 (10) ◽  
pp. E1833-E1839 ◽  
Author(s):  
Andrea Soranno ◽  
Andrea Holla ◽  
Fabian Dingfelder ◽  
Daniel Nettels ◽  
Dmitrii E. Makarov ◽  
...  
Keyword(s):  
Internal Friction ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Experimental Techniques ◽  
Disordered Proteins ◽  
Water Model ◽  
Loop Formation ◽  
Unfolded Proteins ◽  
Intrinsically Disordered ◽  
Unfolded State

Internal friction is an important contribution to protein dynamics at all stages along the folding reaction. Even in unfolded and intrinsically disordered proteins, internal friction has a large influence, as demonstrated with several experimental techniques and in simulations. However, these methods probe different facets of internal friction and have been applied to disparate molecular systems, raising questions regarding the compatibility of the results. To obtain an integrated view, we apply here the combination of two complementary experimental techniques, simulations, and theory to the same system: unfolded protein L. We use single-molecule Förster resonance energy transfer (FRET) to measure the global reconfiguration dynamics of the chain, and photoinduced electron transfer (PET), a contact-based method, to quantify the rate of loop formation between two residues. This combination enables us to probe unfolded-state dynamics on different length scales, corresponding to different parts of the intramolecular distance distribution. Both FRET and PET measurements show that internal friction dominates unfolded-state dynamics at low denaturant concentration, and the results are in remarkable agreement with recent large-scale molecular dynamics simulations using a new water model. The simulations indicate that intrachain interactions and dihedral angle rotation correlate with the presence of internal friction, and theoretical models of polymer dynamics provide a framework for interrelating the contribution of internal friction observed in the two types of experiments and in the simulations. The combined results thus provide a coherent and quantitative picture of internal friction in unfolded proteins that could not be attained from the individual techniques.

scholarly journals Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

2020 ◽  
Author(s):  
Gregory-Neal W. Gomes ◽  
Mickaël Krzeminski ◽  
Ashley Namini ◽  
Erik. W. Martin ◽  
Tanja Mittag ◽  
...  
Keyword(s):  
Experimental Data ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Disordered Proteins ◽  
Saxs Data ◽  
Conformational Ensembles ◽  
Intrinsically Disordered

AbstractIntrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging, but of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We have used an integrative modelling approach to understand how conformational restraints imposed by the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET) reach concordance on structural ensembles for Sic1 and phosphorylated Sic1 (pSic1). To resolve apparent discrepancies between smFRET and SAXS, we integrated SAXS data with non-smFRET (NMR) data and reserved the new smFRET data for Sic1 and pSic1 as an independent validation. The consistency of the SAXS/NMR restrained ensembles with smFRET, which was not guaranteed a priori, indicates that the perturbative effects of NMR or smFRET labels on the Sic1 and pSic1 ensembles are minimal. Furthermore, the mutual agreement with such a diverse set of experimental data suggest that details of the generated ensembles can now be examined with a high degree of confidence to reveal distinguishing features of Sic1 vs. pSic1. From the experimentally well supported ensembles, we find they are consistent with independent biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modelling in calculating and drawing biological conclusions from IDP conformational ensembles.

scholarly journals Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

2019 ◽  
Vol 116 (18) ◽  
pp. 8889-8894 ◽  
Author(s):  
Joshua A. Riback ◽  
Micayla A. Bowman ◽  
Adam M. Zmyslowski ◽  
Kevin W. Plaxco ◽  
Patricia L. Clark ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Analysis Procedure ◽  
Disordered Proteins ◽  
Radius Of Gyration ◽  
Unfolded Proteins ◽  
Intrinsically Disordered ◽  
End Distance ◽  
Polypeptide Chains

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are “uncoupled” (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

scholarly journals Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS

2016 ◽  
Vol 113 (37) ◽  
pp. E5389-E5398 ◽  
Author(s):  
Mikayel Aznauryan ◽  
Leonildo Delgado ◽  
Andrea Soranno ◽  
Daniel Nettels ◽  
Jie-rong Huang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Local Level ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Disordered Proteins ◽  
Time Range ◽  
Unfolded Proteins ◽  
Intrinsically Disordered ◽  
Unfolded State

The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

scholarly journals Exploring Curated Conformational Ensembles of Intrinsically Disordered Proteins in the Protein Ensemble Database

2021 ◽  
Vol 1 (7) ◽  
Author(s):  
Federica Quaglia ◽  
Tamas Lazar ◽  
András Hatos ◽  
Peter Tompa ◽  
Damiano Piovesan ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Conformational Ensembles ◽  
Intrinsically Disordered

scholarly journals The Mysterious Unfoldome: Structureless, Underappreciated, Yet Vital Part of Any Given Proteome

2010 ◽  
Vol 2010 ◽  
pp. 1-14 ◽  
Author(s):  
Vladimir N. Uversky
Keyword(s):  
Large Scale ◽  
Intrinsically Disordered Proteins ◽  
Biologically Active ◽  
Disordered Proteins ◽  
Unfolded Proteins ◽  
Intrinsically Disordered ◽  
Proteome Level ◽  
Natively Unfolded ◽  
Natively Unfolded Proteins

Contrarily to the general believe, many biologically active proteins lack stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) are highly abundant in nature and many of them are associated with various human diseases. The functional repertoire of IDPs complements the functions of ordered proteins. Since IDPs constitute a significant portion of any given proteome, they can be combined in an unfoldome; which is a portion of the proteome including all IDPs (also known as natively unfolded proteins, therefore, unfoldome), and describing their functions, structures, interactions, evolution, and so forth. Amino acid sequence and compositions of IDPs are very different from those of ordered proteins, making possible reliable identification of IDPs at the proteome level by various computational means. Furthermore, IDPs possess a number of unique structural properties and are characterized by a peculiar conformational behavior, including their high stability against low pH and high temperature and their structural indifference toward the unfolding by strong denaturants. These peculiarities were shown to be useful for elaboration of the experimental techniques for the large-scale identification of IDPs in various organisms. Some of the computational and experimental tools for the unfoldome discovery are discussed in this review.

scholarly journals Planar Boronic Graphene and Nitrogenized Graphene Heterostructure for Protein Stretch and Confinement

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1756
Author(s):  
Xuchang Su ◽  
Zhi He ◽  
Lijun Meng ◽  
Hong Liang ◽  
Ruhong Zhou
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Electron Tunneling ◽  
Amyloid Β ◽  
Protein Sequencing ◽  
Disordered Proteins ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Atomic Force ◽  
Dynamics Simulations

Single-molecule techniques such as electron tunneling and atomic force microscopy have attracted growing interests in protein sequencing. For these methods, it is critical to refine and stabilize the protein sample to a “suitable mode” before applying a high-fidelity measurement. Here, we show that a planar heterostructure comprising boronic graphene (BC3) and nitrogenized graphene (C3N) sandwiched stripe (BC3/C3N/BC3) is capable of the effective stretching and confinement of three types of intrinsically disordered proteins (IDPs), including amyloid-β (1–42), polyglutamine (Q42), and α-Synuclein (61–95). Our molecular dynamics simulations demonstrate that the protein molecules interact more strongly with the C3N stripe than the BC3 one, which leads to their capture, elongation, and confinement along the center C3N stripe of the heterostructure. The conformational fluctuations of IDPs are substantially reduced after being stretched. This design may serve as a platform for single-molecule protein analysis with reduced thermal noise.

Molecular simulations of protein disorderThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (2) ◽  
pp. 269-290 ◽  
Author(s):  
Sarah Rauscher ◽  
Régis Pomès
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Peer Review Process ◽  
Structural Heterogeneity ◽  
Disordered Proteins ◽  
Cellular Biology ◽  
Protein Disorder ◽  
Conformational Ensembles ◽  
Intrinsically Disordered ◽  
Simulation Techniques ◽  
Selection Of

Protein disorder is abundant in proteomes throughout all kingdoms of life and serves many biologically important roles. Disordered states of proteins are challenging to study experimentally due to their structural heterogeneity and tendency to aggregate. Computer simulations, which are not impeded by these properties, have recently emerged as a useful tool to characterize the conformational ensembles of intrinsically disordered proteins. In this review, we provide a survey of computational studies of protein disorder with an emphasis on the interdisciplinary nature of these studies. The application of simulation techniques to the study of disordered states is described in the context of experimental and bioinformatics approaches. Experimental data can be incorporated into simulations, and simulations can provide predictions for experiment. In this way, simulations have been integrated into the existing methodologies for the study of disordered state ensembles. We provide recent examples of simulations of disordered states from the literature and our own work. Throughout the review, we emphasize important predictions and biophysical understanding made possible through the use of simulations. This review is intended as both an overview and a guide for structural biologists and theoretical biophysicists seeking accurate, atomic-level descriptions of disordered state ensembles.

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