Structure and Dynamics of an Unfolded Protein Examined by Molecular Dynamics Simulation

174

247

For disordered proteins, the dimensions of the chain are an important property that is sensitive to environmental conditions. We have used single-molecule Förster resonance energy transfer to probe the temperature-induced chain collapse of five unfolded or intrinsically disordered proteins. Because this behavior is sensitive to the details of intrachain and chain-solvent interactions, the collapse allows us to probe the physical interactions governing the dimensions of disordered proteins. We find that each of the proteins undergoes a collapse with increasing temperature, with the most hydrophobic one, λ-repressor, undergoing a reexpansion at the highest temperatures. Although such a collapse might be expected due to the temperature dependence of the classical "hydrophobic effect," remarkably we find that the largest collapse occurs for the most hydrophilic, charged sequences. Using a combination of theory and simulation, we show that this result can be rationalized in terms of the temperature-dependent solvation free energies of the constituent amino acids, with the solvation properties of the most hydrophilic residues playing a large part in determining the collapse.T he properties of unfolded proteins have recently attracted renewed interest (1), triggered in particular by the realization that a large fraction of naturally occurring polypeptides are unstructured under physiological conditions (2, 3). Some of them fold into well-defined structures upon interaction with a ligand or binding partner, whereas others may remain unstructured under all conditions. Many of these "intrinsically disordered proteins" (IDPs) are involved in cellular signaling networks and are thus of great medical interest (4). Given the presence of varying degrees of disorder in unbound and bound states (5), a general framework for the description of the physicochemical properties of IDPs will aid our understanding of the molecular mechanisms underlying their function. Such a framework is beginning to emerge from recent work in which concepts from polymer physics have been found to capture very successfully key aspects of the global conformational and dynamic properties of IDPs and unfolded proteins in general (6). These include the role of charge interactions (7, 8), protein-solvent interactions (9-13), scaling laws (14-16), reconfiguration dynamics (17), and the effect of internal friction (18)(19)(20)(21).An aspect that is less well understood is the effect of temperature on unfolded and intrinsically disordered proteins. Recent single-molecule Förster resonance energy transfer (FRET) experiments showed that the small cold shock protein from Thermotoga maritima (CspTm) and the IDP prothymosin α (ProTα) become more compact with increasing temperature (22), even after the effect of denaturant present in solution (23) is taken into account. The results were in good agreement with dynamic light-scattering experiments on unlabeled protein, demonstrating that the effect is independent of the presence of the fluorophores (22). This result is...

Section: Resultsmentioning

confidence: 99%

Temperature-dependent solvation modulates the dimensions of disordered proteins

Wuttke

Hofmann

Nettels

et al. 2014

174

247

“…These include the immense computer power required to reach the time scale at which foldon behavior emerges, the great accuracy required for the force fields used, and the need for a method that can extract a descriptive trace of the progression of the folding reaction. Recent advances in molecular dynamics trajectory analysis have transcended these limitations in part, bridged the divide between the micro-and macroscopic views of protein folding, and demonstrated that thermally driven amino acid-level searching leads to the native structure through the formation of native-like structural elements and their sequential incorporation in repeatable folding pathways (36)(37)(38). A recent approach explicitly uses foldon conservation and sequential stabilization in an iterative search strategy (39).…”

Section: Discussionmentioning

confidence: 99%

“…The unfolding and refolding of cooperative foldon units and their role in forming intermediates and pathways has been detected, in greater or lesser detail, in many proteins by HX methods (23-25, 29, 47-55), by sulfhydryl labeling (56,57), by relaxation dispersion NMR (58), and in theoretical simulations (36)(37)(38). The inherent cooperativity of native-like foldon units predisposes them rather than other fractional elements to account for the unit steps in protein folding and thus dictates the stepwise formation and native-like nature of pathway intermediates.…”

Section: Discussionmentioning

confidence: 99%

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Hu¹,

Walters

Kan³

et al. 2013

164

217

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on nativelike interfoldon interactions orders the pathway.D o proteins fold through varied and multiple tracks, or do they fold through predetermined intermediates according to understandable biophysical principles (1)? This question is fundamental for the interpretation of a large amount of biophysical and biological research. The question could be resolved if it were possible to define the intermediate structures and pathways that unfolded proteins move through on their way to the native state. Unfortunately, transient intermediates cannot be studied by the usual crystallographic and NMR methods. The range of kinetic and spectroscopic methods has been applied to many proteins, but these methods do not yield the necessary structural information.We used a developing technology, hydrogen exchange pulse labeling measured by MS (HX MS), to study the folding of a cysteine-free variant of Escherichia coli ribonuclease H1 (RNase H), a mixed α/β protein that has served as a major proteinfolding model (2-5). Previous studies showed that RNase H folds in a fast, unresolved burst phase (15 ms dead time) to an intermediate termed "I core " and then much more slowly (in seconds) to the native state (3). HX pulse-labeling and equilibrium native-state HX experiments monitored by NMR showed that I core comprises a continuous region of the protein between helix A and strand 5 and that β-strands 1, 2, and 3 and helix E acquire protection much later, consistent with mutational analysis (2-4). Single-molecule and mutational studies indicated that the intermediate is obligatory, on-pathway, and folds first even when I core is not observably populated (6, 7).The HX MS technique used here is able to follow the entire folding trajectory of RNase H in considerable structural and temporal detail. The analysis monitors every amide site, evaluates the folding coopera...

“…However, a higher-resolution view provided by experimental models (3)(4)(5)(6) and simulations (7) shows that the conformational ensemble is biased toward low-contact-order (CO) structures, e.g., α-helices, β-turns, and β-hairpins, which form and melt in less than a few microseconds. During folding, these nascent structures presumably coalesce into higher-order assemblies of ever-increasing free energy until reaching the transition-state ensemble (TSE) that leads to the native conformation.…”

mentioning

confidence: 99%

Modulation of frustration in folding by sequence permutation

Nobrega

Arora

Kathuria

et al. 2014

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an offpathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N-and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.CF-SAXS | Go models | CheY permutants | protein-folding intermediates H ighly denatured states of globular proteins resemble statistical random coils when examined with low-resolution techniques such as X-ray scattering (1) and hydrodynamic analyses (2). However, a higher-resolution view provided by experimental models (3-6) and simulations (7) shows that the conformational ensemble is biased toward low-contact-order (CO) structures, e.g., α-helices, β-turns, and β-hairpins, which form and melt in less than a few microseconds. During folding, these nascent structures presumably coalesce into higher-order assemblies of ever-increasing free energy until reaching the transition-state ensemble (TSE) that leads to the native conformation. From another perspective, this assembly process mediates a global collapse of the chain in an unfavorable solvent (8). Landscape theory (9) posits that, in the simplest scenario, native-like substructures appear and lead without pause to the TSE and the native conformation in an apparent two-state fashion. However, simulations have found that topological frustration, e.g., the premature formation of a substructure that impedes access to the productive TSE, can lead to the accumulation of intermediates (I) that must unfold to some extent to traverse the folding reaction coordinate...