Abstract
Studying the conformational landscape of intrinsically disordered and partially folded proteins is challenging and only accessible to a few solution state techniques, such as nuclear magnetic resonance (NMR), small-angle scattering techniques, and single-molecule Förster resonance energy transfer (smFRET). While each of the techniques is sensitive to different properties of the disordered chain, such as local structural propensities, overall dimension, or intermediate- and long-range contacts, conformational ensembles describing intrinsically disordered proteins (IDPs) accurately should ideally respect all of these properties. Here we develop an integrated approach using a large set of FRET efficiencies and fluorescence lifetimes, NMR chemical shifts, and paramagnetic relaxation enhancements (PREs), as well as small-angle X-ray scattering (SAXS) to derive quantitative conformational ensembles in agreement with all parameters. Our approach is tested using simulated data (five sets of PREs and 15 FRET efficiencies) and validated experimentally on the example of the disordered domain of measles virus phosphoprotein, providing new insights into the conformational landscape of this viral protein that comprises transient structural elements and is more compact than an unfolded chain throughout its length. Rigorous cross-validation using FRET efficiencies, fluorescence lifetimes, and SAXS demonstrates the predictive nature of the calculated conformational ensembles and underlines the potential of this strategy in integrative dynamic structural biology.
Highlights
IntroductionDisordered proteins (IDPs) play important roles in many biological systems and exert their tasks thanks to their ability to sample conformational ensembles that can have different degrees of compactness and that often comprise transiently folded regions functioning as interaction sites.[1,2] Intrinsically disordered proteins (IDPs) are known to be devoid of stable secondary and tertiary structures, primary structure determines their function and modulates the conformations sampled on a rapid time scale: small motifs can locally enrich the IDP in hydrophobic amino acids, and clusters of charged residues may lead to selfrepulsion, affecting the properties of the chain.[3−5]Single-molecule Förster resonance energy transfer (smFRET) has demonstrated to be a very powerful tool to access the dimension of the unfolded chain through the measurement of energy transfer between site- attached donor and acceptor fluorophores as a function of their distance.[6,7] The technique is compatible with very large IDPs,[8] covering distances that range from 2 to 10 nm approximately, and structural information can be obtained in the presence of transiently folded or folded domains,[9] in complex environments, and even within the living cell.[10,11] Obtaining quantitative structural insight has, remained challenging in particular as the distance between the fluorophores, rather than between their attachment points in the protein backbone, is determined experimentally, and the chemical composition of the dyes and their linkers has to be taken into account in structural modeling
In order to determine conformational ensembles based on experimental Single-molecule Förster resonance energy transfer (smFRET) data, we build on an approach that has been developed and frequently used for calculating conformational ensembles based on diverse nuclear magnetic resonance (NMR) parameters and small-angle X-ray scattering (SAXS).[1,31−34] A large ensemble of conformers (e.g., 10 000) is calculated based on a statistical distribution of Φ and Ψ angles of the protein backbone using the software flexible-meccano.[29]
A molecular description of the conformational landscape sampled by Intrinsically disordered proteins (IDPs) and proteins containing intrinsically disordered regions (IDRs) is of paramount interest, as IDPs and IDRs are enriched in several essential biological processes, such as signaling,[49,50] cellular transport processes,[51,52] and gene regulation,[53,54] and their misregulation is often linked to disease.[55]
Summary
Disordered proteins (IDPs) play important roles in many biological systems and exert their tasks thanks to their ability to sample conformational ensembles that can have different degrees of compactness and that often comprise transiently folded regions functioning as interaction sites.[1,2] IDPs are known to be devoid of stable secondary and tertiary structures, primary structure determines their function and modulates the conformations sampled on a rapid time scale: small motifs can locally enrich the IDP in hydrophobic amino acids, and clusters of charged residues may lead to selfrepulsion, affecting the properties of the chain.[3−5]Single-molecule Förster resonance energy transfer (smFRET) has demonstrated to be a very powerful tool to access the dimension of the unfolded chain through the measurement of energy transfer between site- attached donor and acceptor fluorophores as a function of their distance.[6,7] The technique is compatible with very large IDPs,[8] covering distances that range from 2 to 10 nm approximately, and structural information can be obtained in the presence of transiently folded or folded domains,[9] in complex environments, and even within the living cell.[10,11] Obtaining quantitative structural insight has, remained challenging in particular as the distance between the fluorophores, rather than between their attachment points in the protein backbone, is determined experimentally, and the chemical composition of the dyes and their linkers has to be taken into account in structural modeling. Disordered proteins (IDPs) play important roles in many biological systems and exert their tasks thanks to their ability to sample conformational ensembles that can have different degrees of compactness and that often comprise transiently folded regions functioning as interaction sites.[1,2] IDPs are known to be devoid of stable secondary and tertiary structures, primary structure determines their function and modulates the conformations sampled on a rapid time scale: small motifs can locally enrich the IDP in hydrophobic amino acids, and clusters of charged residues may lead to selfrepulsion, affecting the properties of the chain.[3−5]. These distributions can be expressed as a function of the number of amino acids between the attachment points of the fluorophores, and in order to consider the contribution of the fluorophores and their linkers to the measured distance, they are usually assumed to contribute a number of additional
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