Protein folding mechanisms are probed experimentally using single-point mutant perturbations. The relative effects on the folding ( ϕ -values) and unfolding (1 - ϕ ) rates are used to infer the detailed structure of the transition-state ensemble (TSE). Here we analyze kinetic data on > 800 mutations carried out for 24 proteins with simple kinetic behavior. We find two surprising results: ( i ) all mutant effects are described by the equation: . Therefore all data are consistent with a single ϕ -value (0.24) with accuracy comparable to experimental precision, suggesting that the structural information in conventional ϕ -values is low. ( ii ) ϕ -values change with stability, increasing in mean value and spread from native to unfolding conditions, and thus cannot be interpreted without proper normalization. We eliminate stability effects calculating the ϕ -values at the mutant denaturation midpoints; i.e., conditions of zero stability ( ϕ ⁰ ). We then show that the intrinsic variability is ϕ ⁰ = 0.36 ± 0.11, being somewhat larger for β-sheet-rich proteins than for α-helical proteins. Importantly, we discover that ϕ ⁰ -values are proportional to how many of the residues surrounding the mutated site are local in sequence. High ϕ ⁰ -values correspond to protein surface sites, which have few nonlocal neighbors, whereas core residues with many tertiary interactions produce the lowest ϕ ⁰ -values. These results suggest a general mechanism in which the TSE at zero stability is a broad conformational ensemble stabilized by local interactions and without specific tertiary interactions, reconciling ϕ -values with many other empirical observations.

A large body of work has gone into understanding the effect of mutations on protein structure and function. Conventional treatments have involved quantifying the change in stability, activity and relaxation rates of the mutants with respect to the wild-type protein. However, it is now becoming increasingly apparent that mutational perturbations consistently modulate the packing and dynamics of a significant fraction of protein residues, even those that are located >10-15 Å from the mutated site. Such long-range modulation of protein features can distinctly tune protein stability and the native conformational ensemble contributing to allosteric modulation of function. In this review, I summarize a series of experimental and computational observations that highlight the incredibly pliable nature of proteins and their response to mutational perturbations manifested via the intra-protein interaction network. I highlight how an intimate understanding of mutational effects could pave the way for integrating stability, folding, cooperativity and even allostery within a single physical framework.

The small helical protein BBL has been shown to fold and unfold in the absence of a free energy barrier according to a battery of quantitative criteria in equilibrium experiments, including probe-dependent equilibrium unfolding, complex coupling between denaturing agents, characteristic DSC thermogram, gradual melting of secondary structure, and heterogeneous atom-by-atom unfolding behaviors spanning the entire unfolding process. Here, we present the results of nanosecond T-jump experiments probing backbone structure by IR and end-to-end distance by FRET. The folding dynamics observed with these two probes are both exponential with common relaxation times but have large differences in amplitude following their probe-dependent equilibrium unfolding. The quantitative analysis of amplitude and relaxation time data for both probes shows that BBL folding dynamics are fully consistent with the one-state folding scenario and incompatible with alternative models involving one or several barrier crossing events. At 333 K, the relaxation time for BBL is 1.3 mu s, in agreement with previous folding speed limit estimates. However, late folding events at room temperature are an order of magnitude slower ( 20 mu s), indicating a relatively rough underlying energy landscape. Our results in BBL expose the dynamic features of one-state folding and chart the intrinsic time-scales for conformational motions along the folding process. Interestingly, the simple self-averaging folding dynamics of BBL are the exact dynamic properties required in molecular rheostats, thus supporting a biological role for one-state folding.