Formation of Native and Non-native Interactions in Ensembles of Denatured ACBP Molecules from Paramagnetic Relaxation Enhancement Studies

Kristjánsdóttir, Sigríður; Lindorff‐Larsen, Kresten; Fieber, Wolfgang; Dobson, Christopher M.; Vendruscolo, Michele; Poulsen, Flemming M.

doi:10.1016/j.jmb.2005.01.009

Cited by 88 publications

(164 citation statements)

References 32 publications

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“…Following the work by Gillespie and Shortle, much effort was put into modeling of hr À6 IS i for structural ensembles representing unfolded proteins and predicting the relevant probability distributions. 30,[38][39][40] While the Gillespie-Shortle model is, perhaps, relevant for the case of the compact molten globule with reduced internal mobility (approaching the folded protein limit), it does not fit very well the case where the protein is strongly disordered and behaves essentially as a random coil. Indeed, in this case, the relative motion of the paramagnetic center and the amide 1 H N is best described as relative translation and the conventional formulas for rotational relaxation do not apply.…”

Section: Introductionmentioning

confidence: 99%

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1 H N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1 H N residue number and static magnetic field strength; the results are appreciably different from the Gillespie-Shortle model. At the same time, the Gillespie-Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1 H N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme-using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates-lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent.Additional Supporting Information may be found in the online version of this article.Abbreviations: drkN SH3, N-terminal SH3 domain of the Drosophila adapter protein drk; EDTA, ethylenediaminetetraacetic acid; ESR, electron spin resonance; FRET, fluorescence resonance energy transfer; MTSL, methanethiosulfonate spin label; NOE, nuclear Overhauser effect; NAG, N-acetyl-glycine.

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Section: Introductionmentioning

confidence: 99%

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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“…NMR data measured in disordered proteins have been used as constraints in restrained molecular dynamics or Monte Carlo simulations to direct conformational sampling so that the ensemble is in agreement with experiment (21,22). Alternative approaches have been to filter extensive sampling of unfolded states by proposing weighted populations of conformers to match NMR data (23) or compare averaged experimental data with expected distributions in dihedral angle ( ͞ ) space (24,25).…”

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A structural model for unfolded proteins from residual dipolar couplings and small-angle x-ray scattering

Bernadó

Blanchard

Timmins

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

422

545

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Natively unfolded proteins play key roles in normal and pathological biochemical processes. Despite their importance for function, this category of proteins remains beyond the reach of classical structural biology because of their inherent conformational heterogeneity. We present a description of the intrinsic conformational sampling of unfolded proteins based on residue-specific ͞ propensities from loop regions of a folded protein database and simple volume exclusion. This approach is used to propose a structural model of the 57-aa, natively disordered region of the nucleocapsid-binding domain of Sendai virus phosphoprotein. Structural ensembles obeying these simple rules of conformational sampling are used to simulate averaged residual dipolar couplings (RDCs) and small-angle x-ray scattering data. This protein is particularly informative because RDC data from the equally sized folded and unfolded domains both report on the unstructured region, allowing a quantitative analysis of the degree of order present in this part of the protein. Close agreement between experimental and simulated RDC and small-angle x-ray scattering data validates this simple model of conformational sampling, providing a precise description of local structure and dynamics and average dimensions of the ensemble of sampled structures. RDC data from two urea-unfolded systems are also closely reproduced. The demonstration that conformational behavior of unfolded proteins can be accurately predicted from the primary sequence by using a simple set of rules has important consequences for our understanding of the structure and dynamics of the unstructured state.

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“…protein folding | protein stability | ϕ-value analysis | double-mutant cycle T he properties of nonnative states of proteins have attracted considerable attention because they can play critical roles in cell signaling, translocation across membranes, temperaturesensitive phenotypes, and in a wide range of protein deposition diseases (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The properties of unfolded states are also relevant to any discussion of intrinsically disordered proteins (IDPs) (1).…”

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Energetically significant networks of coupled interactions within an unfolded protein

Cho

Meng

Sato

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pK a measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.protein folding | protein stability | ϕ-value analysis | double-mutant cycle T he properties of nonnative states of proteins have attracted considerable attention because they can play critical roles in cell signaling, translocation across membranes, temperaturesensitive phenotypes, and in a wide range of protein deposition diseases (1-14). The properties of unfolded states are also relevant to any discussion of intrinsically disordered proteins (IDPs) (1). The denatured state ensemble (DSE) can be populated under strongly denaturing conditions and studied at equilibrium; however, the physiologically relevant DSE is the state that is in equilibrium with the folded state under native conditions. This state is difficult to study directly and most studies of the DSE under native conditions have made use of indirect approaches. Nevertheless, experimental and theoretical work has shown that the DSE populated under physiological conditions is not a random coil, but can contain native and nonnative structure, stabilized by local and nonlocal interactions. However, very little is known about the energetics of these interactions, particularly ones involving residues distant in sequence, or about energetically coupled interactions involving multiple residues in the . These are a critical piece of the folding puzzle; they impact protein aggregation and folding, and have important implications for the properties of IDPs. Recent studies offer evidence for hydrophobic clusters, electrostatic interactio...

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Formation of Native and Non-native Interactions in Ensembles of Denatured ACBP Molecules from Paramagnetic Relaxation Enhancement Studies

Cited by 88 publications

References 32 publications

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

A structural model for unfolded proteins from residual dipolar couplings and small-angle x-ray scattering

Energetically significant networks of coupled interactions within an unfolded protein

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