Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues

Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended Muñoz–Eaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and folding–unfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined.     

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than ≈100 μs and distances greater than ≈2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-μs decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type (“D_phys”). D_phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the “speed limit” of folding. D_phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.     

Integrating folding kinetics and protein function: biphasic kinetics and dual binding specificity in a WW domain

Because of the association of beta-sheet formation with the initiation and propagation of amyloid diseases, model systems have been sought to further our understanding of this process. WW domains have been proposed as one such model system. Whereas the folding of the WW domains from human Yes-associated protein (YAP) and Pin have been shown to obey single-exponential kinetics, the folding of the WW domain from formin-binding protein (FBP) 28 has been shown to proceed via biphasic kinetics. From an analysis of free-energy landscapes from atomic-level molecular dynamics simulations, the biphasic folding kinetics observed in the FBP WW domain may be traced to the ability of this WW domain to adopt two slightly different forms of packing in its hydrophobic core. This conformational change is propagated along the peptide backbone and affects the position of a tryptophan residue shown in other WW domains to play a key role in binding. The WW domains of Pin and YAP do not support more than one type of packing each, leading to monophasic folding kinetics. The ability of the FBP WW domain to assume two different types of packing may, in turn, explain the capacity of this WW domain to bind two classes of ligand, a property that is not shared by other WW domains. These findings lead to the hypothesis that lability with respect to conformations separated by an observable barrier as a requirement for function is incompatible with the ability of a protein to fold via single-exponential kinetics.     

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein–solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.Proteins explore many conformations while carrying out their functions in biological systems (1–3). X-ray crystallography is the dominant source of information about protein structure; however, crystal structure models usually consist of just a single major conformation and at most a small portion of the model as alternate conformations. Crystal structures therefore are missing many details about the underlying conformational ensemble (4).Proteins assembled in crystalline arrays, like proteins in solution, exhibit rich conformational diversity (4) and often can perform their native functions (5). Many methods have emerged for using Bragg data to model conformational diversity in protein crystals (6–17). The development of these methods has been important as conformational diversity can lead to inaccuracies in protein structure models (9, 18–20). A key limitation of using the Bragg data, however, is that different models of conformational diversity can yield the same mean electron density.Whereas the Bragg scattering only contains information about the mean electron density, diffuse scattering (diffraction resulting in intensity between the Bragg peaks) is sensitive to spatial correlations in electron density variations (21–28) and therefore contains information about the way that atomic positions vary together in protein crystals. Because models that yield the same mean electron density can yield different correlations in electron density variations, diffuse scattering provides a means to increase the accuracy of crystallography for determining protein conformational variations (29). Peter Moore (30) and Mark Wilson (31) have argued that diffuse scattering should be used to test models of conformational diversity in X-ray crystallography.Several pioneering studies used diffuse scattering to reveal insights into correlated motions in proteins (17, 30, 32–49). Some of these studies used diffuse scattering to experimentally validate predictions of correlated motions from molecular-dynamics (MD) simulations (35–37, 40, 42–44). These studies revealed important insights but were limited by inadequate sampling of the conformational ensemble, leading to lack of convergence of the diffuse scattering calculations (35). Microsecond-scale simulations of staphylococcal nuclease were predicted to be adequate for convergence of diffuse scattering calculations (42). Modern simulation algorithms and computer hardware now enable microsecond or longer MD simulations of protein crystals (50).Here, we present calculations of diffuse X-ray scattering using a 1.1-μs MD simulation of crystalline staphylococcal nuclease. The results demonstrate that we have overcome the past limitation of inadequate sampling. We chose staphylococcal nuclease because the experiments of Wall et al. (49) still represent the only complete, high-quality, 3D diffuse scattering data set from a protein crystal. The calculated diffuse intensity is very similar using two independent halves of the trajectory; the results therefore are reproducible and can be meaningfully compared with the experimental data. The MD simulation provides a rich picture of conformational diversity in the energy landscape of a protein crystal, consisting of at least eight metastable states. Like previous MD studies of crystalline staphylococcal nuclease (42–44), the agreement of the simulation with the total experimental diffuse intensity is excellent, supporting the use of MD simulations to model diffuse scattering data. Unlike previous MD studies, we separately compared the more finely structured, anisotropic component of the diffuse intensity with experimental data. The agreement is substantial but weaker than for the isotropic component, indicating there are inaccuracies in the MD models. Our results therefore point toward using diffuse scattering to improve MD models of protein motions.     

Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein

Protein–protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state. Here we examine a typical system, where a small disordered protein (PUMA, p53 upregulated modulator of apoptosis) folds to an α-helix when bound to a groove on the surface of a folded protein (MCL-1, induced myeloid leukemia cell differentiation protein). We follow the association of these proteins using rapid-mixing stopped flow, and examine how the kinetic behavior is perturbed by denaturant and carefully chosen mutations. We demonstrate the utility of methods developed for the study of monomeric protein folding, including β-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.For many proteins, correct folding to a specific 3D structure is essential for their function inside the cell; once folded, some of these have the appropriate shape and accessible chemical groups to interact specifically with, and bind to, another protein (1). However, for a number of protein–protein interactions, folding and binding do not appear to be separate, sequential events (2, 3). Many intrinsically disordered proteins (IDPs) will appear largely unfolded in isolation, only forming a specific structure when bound to an appropriate partner protein and undergoing coupled folding and binding (4–6). Such reactions are abundant in signaling and regulatory processes (7, 8). Protein folding does not simply provide correctly shaped building blocks for the cell; it can play an intimate role in molecular recognition.Over the past decade, bioinformatics studies have revealed that protein disorder (7, 9), and coupled folding and binding (10), are widespread in biology. Many structures of bound, folded IDPs have been solved and have shown the wide range of topologies that can be formed (11). Biophysical techniques (12), NMR in particular (13), can characterize isolated IDPs in detail. Despite this progress, the number of studies examining kinetics and the mechanisms of binding remains relatively small (14–21) given that the most commonly observed function of IDPs is in coupled folding and binding reactions (22).To describe coupled folding and binding, two extreme mechanisms are often discussed, focusing on whether an IDP needs to fold before interacting productively with its binding partner. In isolation an IDP could, perhaps only transiently, occupy a conformation that resembles the bound state. In the pure conformational selection mechanism, the IDP must be in this conformation at the start of the eventually successful encounter with the partner protein (23, 24) (Fig. 1A). Arguments in support of this mechanism largely come from NMR studies that have successfully detected these lowly populated, folded states in unbound IDPs (25–27). In the contrasting induced-fit mechanism, there is no requirement for the IDP to fold in isolation (28). Instead, the potentially transient interactions with the partner protein lead to the folding of the IDP (Fig. 1A). Complex mixtures of these two extreme mechanisms can also be imagined: e.g., perhaps only a proportion of the IDP needs to fold before the encounter, i.e., conformational selection followed by induced fit of the remaining peptide chain (29). To add to the potential complexity, flux through different pathways could occur simultaneously, and may depend on the concentrations of protein involved (23, 30). Further, confirming the degree of induced fit and conformational selection is only one aspect of the binding mechanism. There remain a large number of mechanistic possibilities beyond the state of the IDP prior to successful encounters.Open in a separate windowFig. 1.PUMA–MCL-1 binding. (A) Cartoon of binding mechanisms. IDP PUMA (blue) can undergo coupled folding and binding with structured MCL-1 (white) to form a single, contiguous α-helix. Structures based on PDB 2ROC (39) and 1WSX (58). Unbound PUMA and encounter complex built using Chimera (University of California, San Francisco). Figure prepared using PyMol. (B) Representative fluorescence stopped-flow traces for binding. Increasing the concentration of urea from 0 to 3.5 M (in 0.5-M increments) slows association. (C) The urea dependence of the natural log of the association rate constant (k+) for the wild-type PUMA peptide used in this study. (D) The urea dependence of the dissociation rate constant (k−). k− was determined by preforming the PUMA–MCL-1 complex at micromolar concentrations and manually diluting to nanomolar concentrations to induce dissociation. The resulting kinetic trace was fit to a reversible model, fixing k+ from the association experiments (41). Gradient of the linear fits corresponds to the m values discussed in the main text. A, B, and C adapted from ref. 37.It is largely agreed that most protein folding (and unfolding) reactions are limited by the requirement to populate a high-energy transition state (31). Kinetic, time-resolved experiments, in combination with site-directed mutagenesis and Φ-value analysis (32), have been applied successfully to describe these transition states (33, 34). With carefully chosen mutations, the distribution of Φ values (classically between 0 and 1) offers an average picture of the interactions formed at this critical stage of the folding reaction, at residue-level resolution. This picture, in conjunction with other evidence, can offer invaluable insights into the mechanisms of folding (35, 36).We have previously reported the kinetics of a model coupled folding and binding reaction (37, 38); the BH3 motif of PUMA (an IDP) can associate with the structured protein MCL-1 and fold to a single contiguous α-helix (39). The solvent and temperature dependence of the association reaction suggested that this reaction is limited by a free energy barrier, or transition state (TS) (37). Here we systematically make structurally conservative mutations to the IDP and the partner protein, apply Φ-value analysis, and describe the transition state for binding. Molecular dynamics simulations using a coarse-grained, topology-based model of the binding process are consistent with our experimental results. We bring together all available evidence to propose a mechanism of binding.