scholarly journals Abundant α-Synuclein compact dimers

2019 ◽  
Author(s):  
Sofia Zaer ◽  
Paz Drori ◽  
Mario Lebendiker ◽  
Yair Razvag ◽  
Eitan Lerner
Keyword(s):  
Light Scattering ◽  
Single Molecule ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Abundant Species ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Correlation Spectroscopy ◽  
Intrinsically Disordered ◽  
Donor And Acceptor

Abstractα-Synuclein (αSyn) is an intrinsically disordered protein that forms oligomers and fibrils associated with Parkinson’s disease. As such, the mechanism of its oligomerization and its possible links to neurotoxicity have been the focus of many studies. Out of the numerous oligomer types, dimers are the smallest oligomers of aSyn that have been reported. As such, αSyn dimers serve as the earliest steps in the nucleation of αSyn oligomers and later fibrils. Therefore, it is important to characterize αSyn dimers. The identification of αSyn dimers in ensemble-averaged measurements without the use of chemical modifications have been difficult, due to their apparent low abundance. Using analytical anion exchange chromatography coupled to multi angle light scattering as well as to dynamic light scattering, we show that recombinant αSyn is in equilibrium between different types of monomers and compact dimers, and that both are abundant. Additionally, bulk Förster resonance energy transfer (FRET), fluorescence cross-correlation spectroscopy (FCCS) of FRET and pulsed-interleaved excitation single-molecule FRET (PIE smFRET) measurements of mixtures of donor- and acceptor-labeled αSyn. These measurements indicated a dimer dissociation constant of 1.75 μM. We concluded that αSyn dimers exist as abundant species in equilibrium with monomers only if produced to reach concentrations of hundreds of nanomolar or above.

scholarly journals The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

2020 ◽  
Author(s):  
Jiaxing Chen ◽  
Sofia Zaer ◽  
Paz Drori ◽  
Joanna Zamel ◽  
Khalil Joron ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Structural Heterogeneity ◽  
Conformational Space ◽  
Conformational Ensemble ◽  
Time Resolved ◽  
Intrinsically Disordered ◽  
Transient Nature

AbstractThe intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

scholarly journals A functional role for intrinsic disorder in the tau-tubulin complex

2016 ◽  
Vol 113 (50) ◽  
pp. 14336-14341 ◽  
Author(s):  
Ana M. Melo ◽  
Juliana Coraor ◽  
Garrett Alpha-Cobb ◽  
Shana Elbaum-Garfinkle ◽  
Abhinav Nath ◽  
...  
Keyword(s):  
Single Molecule ◽  
Solution Structure ◽  
Dynamic Instability ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Conformational Ensemble ◽  
Binding Region ◽  
Global Features ◽  
Microtubule Binding ◽  
Intrinsically Disordered

Tau is an intrinsically disordered protein with an important role in maintaining the dynamic instability of neuronal microtubules. Despite intensive study, a detailed understanding of the functional mechanism of tau is lacking. Here, we address this deficiency by using intramolecular single-molecule Förster Resonance Energy Transfer (smFRET) to characterize the conformational ensemble of tau bound to soluble tubulin heterodimers. Tau adopts an open conformation on binding tubulin, in which the long-range contacts between both termini and the microtubule binding region that characterize its compact solution structure are diminished. Moreover, the individual repeats within the microtubule binding region that directly interface with tubulin expand to accommodate tubulin binding, despite a lack of extension in the overall dimensions of this region. These results suggest that the disordered nature of tau provides the significant flexibility required to allow for local changes in conformation while preserving global features. The tubulin-associated conformational ensemble is distinct from its aggregation-prone one, highlighting differences between functional and dysfunctional states of tau. Using constraints derived from our measurements, we construct a model of tubulin-bound tau, which draws attention to the importance of the role of tau’s conformational plasticity in function.

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 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 Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

scholarly journals Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

2020 ◽  
Author(s):  
Franziska Zosel ◽  
Andrea Holla ◽  
Benjamin Schuler
Keyword(s):  
Fluorescence Spectroscopy ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Control Sample ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Single Molecule Fluorescence ◽  
Intrinsically Disordered ◽  
Single Molecule Fluorescence Spectroscopy

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. Here we describe the practical details of a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.<br>

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