Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

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.

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Commonly-used FRET fluorophores promote collapse of an otherwise disordered protein

10.1101/376632 ◽

2018 ◽

Cited By ~ 1

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 ◽

Protein Chain ◽

Intrinsically Disordered ◽

Tightly Coupled ◽

End Distance

ABSTRACTThe dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt at low or no denaturant remains controversial. We recently developed an innovative analysis procedure for small-angle X-ray scattering (SAXS) profiles and found that even relatively hydrophobic IDPs remain nearly as expanded as the chemically denatured ensemble, rendering them significantly more expanded than generally inferred using fluorescence resonance energy transfer (FRET) measurements. We show here that fluorophores typical of those employed in FRET can contribute to this discrepancy. Specifically, we find that addition of Alexa488 to a normally expanded IDP causes contraction of its ensemble. In parallel, we also tested the recent suggestion that FRET and SAXS results can be reconciled if, in contrast to homopolymers, the radius of gyration (Rg) of an unfolded protein chain can vary independently from its end-to-end distance (Ree). To do so, we developed an analysis procedure that can accurately extract both Rg and Ree from SAXS profiles even if they are decoupled. Using this procedure, we find that Rg and Ree remain tightly coupled even for heteropolymeric IDPs. We thus conclude that, when combined with improved analysis procedures for both SAXS and FRET, fluorophore-driven interactions are sufficient to explain the preponderance of existing data regarding the nature of polypeptide chains unfolded in the absence of denaturant.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704692114 ◽

2017 ◽

Vol 114 (31) ◽

pp. E6342-E6351 ◽

Cited By ~ 100

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.

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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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Single-molecule spectroscopy of unfolded proteins and chaperonin action

Biological Chemistry ◽

10.1515/hsz-2014-0132 ◽

2014 ◽

Vol 395 (7-8) ◽

pp. 689-698 ◽

Cited By ~ 3

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.

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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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Single-Molecule FRET of Intrinsically Disordered Proteins

Annual Review of Physical Chemistry ◽

10.1146/annurev-physchem-012420-104917 ◽

2020 ◽

Vol 71 (1) ◽

pp. 391-414 ◽

Cited By ~ 4

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607193113 ◽

2016 ◽

Vol 113 (37) ◽

pp. E5389-E5398 ◽

Cited By ~ 84

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.

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

10.1101/2021.01.19.427359 ◽

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.

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

Biomolecules ◽

10.3390/biom12010027 ◽

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.

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

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/568068 ◽

2010 ◽

Vol 2010 ◽

pp. 1-14 ◽

Cited By ~ 146

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.

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