scholarly journals Synergies of Single Molecule Fluorescence and NMR for the Study of Intrinsically Disordered Proteins

Biomolecules ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 27
Author(s):  
Samuel Naudi-Fabra ◽  
Martin Blackledge ◽  
Sigrid Milles
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Resonance Spectroscopy ◽  
Multidomain Proteins ◽  
Single Molecule Fluorescence ◽  
Intrinsically Disordered ◽  
Shed Light

Single molecule fluorescence and nuclear magnetic resonance spectroscopy (NMR) are two very powerful techniques for the analysis of intrinsically disordered proteins (IDPs). Both techniques have individually made major contributions to deciphering the complex properties of IDPs and their interactions, and it has become evident that they can provide very complementary views on the distance-dynamics relationships of IDP systems. We now review the first approaches using both NMR and single molecule fluorescence to decipher the molecular properties of IDPs and their interactions. We shed light on how these two techniques were employed synergistically for multidomain proteins harboring intrinsically disordered linkers, for veritable IDPs, but also for liquid–liquid phase separated systems. Additionally, we provide insights into the first approaches to use single molecule Förster resonance energy transfer (FRET) and NMR for the description of multiconformational models of IDPs.

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>

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

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 140 ◽  
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.

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 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>

Single-Molecule FRET of Intrinsically Disordered Proteins

2020 ◽  
Vol 71 (1) ◽  
pp. 391-414 ◽  
Author(s):  
Lauren Ann Metskas ◽  
Elizabeth Rhoades
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Intrinsically Disordered ◽  
Single Molecule Fret

Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments. This review provides an overview of confocal diffusion-based smFRET as an experimental tool, including descriptions of instrumentation, data analysis, and protein labeling. Recent papers are discussed that illustrate the unique capability of smFRET to provide insight into aggregation-prone IDPs, protein–protein interactions involving IDPs, and IDPs in complex experimental milieus.

scholarly journals Simulating freely-diffusing single-molecule FRET data with consideration of protein conformational dynamics

2021 ◽  
Author(s):  
James Losey ◽  
Michael Jauch ◽  
David S. Matteson ◽  
Mahmoud Moradi
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Analysis Techniques ◽  
Intrinsically Disordered ◽  
Single Molecule Fret ◽  
Great Progress

AbstractSingle molecule Förster resonance energy transfer experiments have added a great deal to the under-standing of conformational states of biologically important molecules. While great progress has been made, much is still unknown in systems that are highly flexible such as intrinsically disordered proteins because of the high degeneracy of distance states, particularly when freely diffusing smFRET experiments are used. Simulated smFRET data allows for the control of underlying process that generates the data to examine if analytic techniques can detect these underlying differences. We have extended the PyBroMo software that simulates the freely diffusing smFRET data to include a distribution of inter-dye distances generated using Langevin dynamics in order to model proteins with greater flexibility or disorder in structure. Standard analysis techniques for smFRET data compared highlighted the differences observed between data generated with the base software and data that included the distribution of inter-dye distance.

scholarly journals Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

2017 ◽  
Vol 114 (31) ◽  
pp. E6342-E6351 ◽  
Author(s):  
Gustavo Fuertes ◽  
Niccolò Banterle ◽  
Kiersten M. Ruff ◽  
Aritra Chowdhury ◽  
Davide Mercadante ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Heterogeneous Systems ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Radius Of Gyration ◽  
Intrinsically Disordered ◽  
High Concentrations ◽  
The Impact

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE. For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE, whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

scholarly journals Recent Advances in Computational Protocols Addressing Intrinsically Disordered Proteins

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 146 ◽  
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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