Atomic-level description of ubiquitin folding

Abstract

Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. CHARMM22* | energy landscape | enthalpy | phi-value analysis | prefactor U nderstanding the principles that govern protein folding, the self-assembly process that leads from an unstructured polypeptide chain to a fully functional protein, has been one of the major challenges in the area of physical biochemistry over the last 50 years. Naturally occurring proteins typically fold on timescales ranging from milliseconds to minutes, but as our understanding of the principles of protein folding has improved, a number of fastfolding proteins that fold on the microsecond timescale have been engineered and characterized (1-12). The design of such fastfolding proteins was in part intended to narrow the gap between the timescales of protein folding and the timescale accessible to physics-based atomistic molecular dynamics (MD) simulations, thus enabling fruitful combinations of experiments and simulations to study the mechanisms of folding (13-18). We recently studied 12 fast-folding proteins using equilibrium MD simulations, for example, with the aim of elucidating general principles underlying the folding of these proteins (19). The folding of these proteins was found to be a relatively sequential process that follows a few paths, in which the order of formation of native structure is correlated with relative structural stability in the unfolded state. The extent to which these observations pertained to fast-folding proteins only or to protein folding more generally, however, was unclear. Here we address this question by applying the same methodology and physics-based force field used in our previous investigation of fast-folding proteins to the study of ubiquitin, a nat...

Folding pathway of a multidomain protein depends on its topology of domain connectivity

“…σ i = 0 when the ith residue was proline, k B σ i = 3 cal·mol −1 ·K −1 when the ith residue was Gly, and k B σ i = 2 cal·mol −1 ·K −1 for other residues (41). Eighty to ninety percent of proline residues are considered to remain in the trans configuration upon unfolding (27,51), and we selected e pro = 1:8 kcal/mol to make this ratio 90% for the sake of computatinal efficiency.…”

Allosteric switch regulates protein–protein binding through collective motion

“…Thus, it is highly likely that the pincer mode moves on a time scale faster than the RD detection limit but slower than the tumbling time of ubiquitin, putting the time scale in the tens to hundreds of nanoseconds. Indeed, the peptide flip motion is also slower than the pincer mode in MD trajectories from two different studies (10,22).…”

“…S8). This observation suggests that although at least two processes occur on the microsecond time scale [peptide flipping and motion around I36 (22)(23)(24)], peptide flipping is directly coupled with the majority of the conformational fluctuation throughout the structure. This finding is further supported by the temperature dependence of 1 H N RD, in which the majority of residues show profiles that coincide with E24 and G53 (Fig.…”

Determining protein structures by combining semireliable data with atomistic physical models by Bayesian inference

“…This may be due to differences in torsion angles; Huber et al (25) used TALOS+ predictions based on NMR data, whereas our X-PLOR calculations used sequence-based predictions. not able to find the structure of ubiquitin starting from an extended chain, because ubiquitin folds only on the millisecond timescale (17). In MELD, however, even though the restraints are sparse, this set is sufficient to provide a highly funneled free-energy landscape (SI Appendix, Fig.…”

“…2, which may alone be enough to correctly predict the structures of small proteins (17)(18)(19). The likelihood function, pðDjxÞ, expresses our belief about how probable it is that the observed data were produced by a particular structure (discussed below).…”