Atom-by-atom analysis of global downhill protein folding

Proceedings of the National Academy of Sciences, 2008

Protein folding barriers, which range from zero to the tens of RT that result in classical two-state kinetics, are primarily determined by protein size and structural topology [Plaxco KW, Simons KT, Baker D (1998) J Mol Biol 277:985-994]. Here, we investigate the thermodynamic folding barriers of two relatively large proteins of the same size and topology: bovine ␣-lactalbumin (BLA) and hen-egg-white lysozyme (HEWL). From the analysis of differential scanning calorimetry experiments with the variable-barrier model [Muñ oz V, Sanchez-Ruiz JM (2004) Proc Natl Acad Sci USA 101: 17646 -17651] we obtain a high barrier for HEWL and a marginal folding barrier for BLA. These results demonstrate a remarkable tuning range of at least 30 kJ/mol (i.e., five to six orders of magnitude in population) within a unique protein scaffold. Experimental and theoretical analyses on these proteins indicate that the surprisingly small thermodynamic folding barrier of BLA arises from the stabilization of partially unfolded conformations by electrostatic interactions. Interestingly, there is clear reciprocity between the barrier height and the biological function of the two proteins, suggesting that the marginal barrier of BLA is a product of natural selection. Electrostatic surface interactions thus emerge as a mechanism for the modulation of folding barriers in response to special functional requirements within a given structural fold.

Structural dynamics (Melville, N.Y.), 2017

This review provides a comprehensive overview of the structural dynamics in topical gas- and condensed-phase systems on multiple length and time scales. Starting from vibrationally induced dissociation of small molecules in the gas phase, the question of vibrational and internal energy redistribution through conformational dynamics is further developed by considering coupled electron/proton transfer in a model peptide over many orders of magnitude. The influence of the surrounding solvent is probed for electron transfer to the solvent in hydrated I. Next, the dynamics of a modified PDZ domain over many time scales is analyzed following activation of a photoswitch. The hydration dynamics around halogenated amino acid side chains and their structural dynamics in proteins are relevant for iodinated TyrB26 insulin. Binding of nitric oxide to myoglobin is a process for which experimental and computational analyses have converged to a common view which connects rebinding time scales and t...

Physical chemistry chemical physics : PCCP, 2017

Transcription factors are thought to efficiently search for their target DNA site via a combination of conventional 3D diffusion and 1D diffusion along the DNA molecule mediated by non-specific electrostatic interactions. This process requires the DNA-binding protein to quickly exchange between a search competent and a target recognition mode, but little is known as to how these two binding modes are encoded in the conformational properties of the protein. Here, we investigate this issue on the engrailed homeodomain (EngHD), a DNA-binding domain that folds ultrafast and exhibits a complex conformational behavior consistent with the downhill folding scenario. We explore the interplay between folding and DNA recognition using a coarse-grained computational model that allows us to manipulate the folding properties of the protein and monitor its non-specific and specific binding to DNA. We find that conformational disorder increases the search efficiency of EngHD by promoting a fast gli...

International Journal of Molecular Sciences

The structural effects of zinc replacement by xenobiotic metal ions have been widely studied in several eukaryotic and prokaryotic zinc-finger-containing proteins. The prokaryotic zinc finger, that presents a bigger βββαα domain with a larger hydrophobic core with respect to its eukaryotic counterpart, represents a valuable model protein to study metal ion interaction with metallo-proteins. Several studies have been conducted on Ros87, the DNA binding domain of the prokaryotic zinc finger Ros, and have demonstrated that the domain appears to structurally tolerate Ni(II), albeit with important structural perturbations, but not Pb(II) and Hg(II), and it is in vitro functional when the zinc ion is replaced by Cd(II). We have previously shown that Ros87 unfolding is a two-step process in which a zinc binding intermediate converts to the native structure thorough a delicate downhill folding transition. Here, we explore the folding/unfolding behaviour of Ros87 coordinated to Co(II), Ni(II...

Physical Chemistry Chemical Physics, 2011

Physical Chemistry Chemical Physics

Using multi-site time-resolved FRET, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the β-sheet.

Scientific Reports, 2020

Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. Downhill folders are estimated to be rather rare in nature as they miss an energetically substantial folding barrier that can protect against aggregation and proteolysis. We have previously demonstrated that the prokaryotic zinc finger protein Ros87 shows a bipartite folding/unfolding process in which a metal binding intermediate converts to the native structure through a delicate barrier-less downhill transition. Significant variation in folding scenarios can be detected within protein families with high sequence identity and very similar folds and for the same sequence by varying conditions. For this reason, we here show, by means of DSC, CD and NMR, that also in different pH and ionic strength conditions Ros87 retains its partly downhill folding scenario demonstrating that, at least in metallo-proteins, t...

The Journal of Chemical Physics, 2008

Previously we presented an all-atom force field with a generalized Born solvation model ͑param99MOD5/GBSA͒ for a more balanced description of ␣ / ␤ propensities. We performed direct folding simulations on ␣ helices ͑PDB code 2I9M and 1WN8͒, ␤ hairpins ͑15␤ peptide and PDB code 1E0Q͒, and ␤-sheet peptide ͑ D P D P-II͒ to investigate the transferability of a new param99MOD5/GBSA force field. For direct folding simulations, we used the replica exchange molecular dynamics simulation starting with a fully extended conformer. In the converged free energy landscapes for all five peptides, each of the lowest free energy predicted structures closely matched the corresponding NMR native structure within a backbone rmsd value of 2.0 Å at experimental temperatures. The thermal denaturation profiles of all the peptides fit a two-state model well, giving several key thermodynamic parameters for comparison. Especially for 15␤ and D P D P-II whose thermodynamic data were available from the experiment, our simulated thermodynamic quantities agree reasonably well with the experiment. In this work, we demonstrate that the modified force field successfully differentiates native structures of ␣ and ␤ strands under the global free energy minimum condition, so that it can be used in ab initio folding simulations for more complex motifs.

Nature communications, 2016

A major drive in protein folding has been to develop experimental technologies to resolve the myriads of microscopic pathways and complex mechanisms that purportedly underlie simple two-state folding behaviour. This is key for cross-validating predictions from theory and modern computer simulations. Detecting such complexity experimentally has remained elusive even using methods with improved time, structural or single-molecule resolution. Here, we investigate the mechanical unfolding of cold shock protein B (Csp), a showcase two-state folder, using single-molecule force-spectroscopy. Under controlled-moderate pulling forces, the unfolding of Csp emerges as highly heterogeneous with trajectories ranging from single sweeps to different combinations of multiple long-lived mechanical intermediates that also vary in order of appearance. Steered molecular dynamics simulations closely reproduce the experimental observations, thus matching unfolding patterns with structural events. Our res...

Physical chemistry chemical physics : PCCP, 2017

Proteins that fold upon binding to their partners exhibit complex binding behavior such as induced-fit. But the connections between the folding mechanism and the binding mode remain unknown. Here we focus on the high affinity complex between the physiologically and marginally unstable, fast folder PSBD and the E1 subunit of pyruvate dehydrogenase. Using coarse-grained simulations we investigate the binding to E1 of a partially disordered PSBD under two folding scenarios: two-state and downhill. Our simulations show that induced-fit binding requires that PSBD folds-unfolds in the downhill folding regime. In contrast, a two-state folding PSBD must fold completely before it binds. The reason is that effective coupling between folding and binding involves partially folded conformations, which are only sufficiently populated under the downhill folding regime. Our results establish a direct mechanistic link between complex binding and downhill folding, supporting the idea that PSBD operat...

Proceedings of the National Academy of Sciences, 2007

Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative (“downhill”) folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at ≥4 × 10 4 s −1 at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same time-averaged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at ...

Proceedings of the National Academy of Sciences, 2009

One controversial area in protein folding mechanisms is whether some small, ultra-fast-folding proteins exist in distinct native and denatured state ensembles, separated by an energy barrier, or if there is a continuum of states between native and denatured. In theory, the simplest way of distinguishing between single-state barrierless or “downhill” folding and conventional separate state folding is by single-molecule spectroscopy, which can detect either distinct populations of proteins or a continuum. But, the time resolution of approximately 1 ms of most confocal fluorescence microscopes for single-molecule fluorescence resonance energy transfer (SM-FRET) is longer than that for the structural relaxation of proteins such as BBL, whose mechanism of folding is controversial. We have constructed a highly sensitive confocal fluorescence microscope and measured the distribution of FRET efficiencies of appropriately labeled BBL in time bins of 50 and 200 μs under conditions in which it...

Proceedings of the National Academy of Sciences, 2008

In contrast to classical chemical phenomenology, theory suggests that proteins may undergo downhill folding without an activation barrier under certain thermodynamic conditions. Recently, the BBL protein was proposed to fold by such a downhill scenario, but discrepancies between experimental results found in different groups argue against this. After briefly reviewing the major experimental studies of the BBL folding mechanism, we show that simulations of both coarse-grained and atomistic models can reconcile the seemingly conflicting observations.

Communications Chemistry, 2018

Ultrafast folding proteins have limited cooperativity and thus are excellent models to resolve, via single-molecule experiments, the fleeting molecular events that proteins undergo during folding. Here we report single-molecule atomic force microscopy experiments on gpW, a protein that, in bulk, folds in a few microseconds over a marginal folding barrier (∼1 kBT). Applying pulling forces of only 5 pN, we maintain gpW in quasi-equilibrium near its mechanical unfolding midpoint and detect how it interconverts stochastically between the folded and an extended state. The interconversion pattern is distinctly binary, indicating that, under an external force, gpW (un)folds over a significant free-energy barrier. Using molecular simulations and a theoretical model we rationalize how force induces such barrier in an otherwise downhill free-energy surface. Force-induced folding barriers are likely a general occurrence for ultrafast folding biomolecules studied with single-molecule force spec...

The Journal of biological chemistry, 2018

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with sub-denaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, likely representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Flu...