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

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1610137113 ◽

2016 ◽

Vol 113 (50) ◽

pp. 14336-14341 ◽

Cited By ~ 33

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.

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

10.1101/2020.02.05.935890 ◽

2020 ◽

Cited By ~ 2

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.

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Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife ◽

10.7554/elife.70090 ◽

2021 ◽

Vol 10 ◽

Author(s):

Abhishek Mazumder ◽

Richard H Ebright ◽

Achillefs Kapanidis

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Transcription Initiation ◽

Core Promoter ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Extensive Study ◽

Active Centre ◽

Transient Nature ◽

Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

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Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025578118 ◽

2021 ◽

Vol 118 (33) ◽

pp. e2025578118

Author(s):

Lena Voith von Voithenberg ◽

Anders Barth ◽

Vanessa Trauschke ◽

Benjamin Demarco ◽

Swati Tyagi ◽

...

Keyword(s):

Single Molecule ◽

Structural Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Conformational Space ◽

Distribution Analysis ◽

Nucleotide Exchange ◽

Nucleotide Analogs ◽

Recognition Mechanism ◽

Substrate Peptide

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

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Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity

10.1101/740241 ◽

2019 ◽

Author(s):

Amberley D. Stephens ◽

Maria Zacharopoulou ◽

Rani Moons ◽

Giuliana Fusco ◽

Neeleema Seetaloo ◽

...

Keyword(s):

Long Range ◽

Intrinsically Disordered Protein ◽

Local Environment ◽

Alpha Synuclein ◽

Conformational Space ◽

Conformational Ensemble ◽

Aggregation Propensity ◽

Intrinsically Disordered ◽

Long Range Interactions ◽

N Terminus

AbstractAs an intrinsically disordered protein, monomeric alpha synuclein (aSyn) constantly reconfigures and probes the conformational space. Long-range interactions across the protein maintain its solubility and mediate this dynamic flexibility, but also provide residual structure. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. By using calcium to perturb the conformational ensemble, we observe differences in structure and intra-molecular dynamics between two aSyn C-terminal variants, D121A and pS129, and the aSyn familial disease mutants, A30P, E46K, H50Q, G51D, A53T and A53E, compared to wild-type (WT) aSyn. We observe that the more exposed the N-terminus and the beginning of the NAC region are, the more aggregation prone monomeric aSyn conformations become. N-terminus exposure occurs upon release of C-terminus interactions when calcium binds, but the level of exposure is specific to the aSyn mutation present. There was no correlation between single charge alterations, calcium affinity, or the number of ions bound on aSyn’s aggregation propensity, indicating that sequence or post-translation modification (PTM)-specific conformational differences between the N- and C-termini and the specific local environment mediate aggregation propensity instead. Understanding aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein, to stabilise aSyn in non-aggregation prone conformations, by either preserving long-range interactions between the N- and C-termini or by protecting the N-terminus from exposure.

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Abundant α-Synuclein compact dimers

10.1101/795997 ◽

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.

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Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins

Biomolecules ◽

10.3390/biom8040140 ◽

2018 ◽

Vol 8 (4) ◽

pp. 140 ◽

Cited By ~ 17

Author(s):

Sharonda LeBlanc ◽

Prakash Kulkarni ◽

Keith Weninger

Keyword(s):

Single Molecule ◽

Biological Networks ◽

Intrinsically Disordered Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Polymer Physics ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Single Molecule Fret ◽

Random Fluctuations

Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.

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Recent Advances in Computational Protocols Addressing Intrinsically Disordered Proteins

Biomolecules ◽

10.3390/biom9040146 ◽

2019 ◽

Vol 9 (4) ◽

pp. 146 ◽

Cited By ~ 9

Author(s):

Supriyo Bhattacharya ◽

Xingcheng Lin

Keyword(s):

Single Molecule ◽

Degrees Of Freedom ◽

Intrinsically Disordered Proteins ◽

Structural Information ◽

Conformational Dynamics ◽

Resonance Energy ◽

Structural Data ◽

Disordered Proteins ◽

Conformational Ensemble ◽

Intrinsically Disordered

Intrinsically disordered proteins (IDP) are abundant in the human genome and have recently emerged as major therapeutic targets for various diseases. Unlike traditional proteins that adopt a definitive structure, IDPs in free solution are disordered and exist as an ensemble of conformations. This enables the IDPs to signal through multiple signaling pathways and serve as scaffolds for multi-protein complexes. The challenge in studying IDPs experimentally stems from their disordered nature. Nuclear magnetic resonance (NMR), circular dichroism, small angle X-ray scattering, and single molecule Förster resonance energy transfer (FRET) can give the local structural information and overall dimension of IDPs, but seldom provide a unified picture of the whole protein. To understand the conformational dynamics of IDPs and how their structural ensembles recognize multiple binding partners and small molecule inhibitors, knowledge-based and physics-based sampling techniques are utilized in-silico, guided by experimental structural data. However, efficient sampling of the IDP conformational ensemble requires traversing the numerous degrees of freedom in the IDP energy landscape, as well as force-fields that accurately model the protein and solvent interactions. In this review, we have provided an overview of the current state of computational methods for studying IDP structure and dynamics and discussed the major challenges faced in this field.

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Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors

npj Parkinson s Disease ◽

10.1038/s41531-021-00195-6 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Anthony R. Braun ◽

Elly E. Liao ◽

Mian Horvath ◽

Prakriti Kalra ◽

Karen Acosta ◽

...

Keyword(s):

Drug Discovery ◽

Single Molecule ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Alpha Synuclein ◽

Time Resolved ◽

Primary Mouse ◽

Fret Biosensors

AbstractWe have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson’s disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

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