Insights into protein folding mechanisms from large scale analysis of mutational effects

Abstract

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: ΔΔG ‡ unfold ¼ 0.76ΔΔG eq AE 1.8 kJ∕mol. 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 ¼ 0.36 AE 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. kinetics | mutations | phi-values | perturbation analysis | free energy relationships P rotein engineering has dominated the experimental study of folding mechanisms for nearly two decades(1-3). In this method, single-point mutations are studied kinetically to determine the relative effects in folding and unfolding rates. The basic approach is based on the common observation that the logarithm of the folding relaxation rate changes with chemical denaturant in a V-shaped fashion (i.e., the chevron plot) (4). For a two-state folding protein the low and high denaturant limbs report on the folding and unfolding rate constants, respectively. Linear chevron limbs are taken to imply that there is a thermodynamically well-defined transition-state ensemble (TSE) with intermediate sensitivity to chemical denaturant (4). Accordingly, the perturbation free energy produced by mutation (ΔΔG eq ) partitions between the folding (ϕ) and unfolding limbs (1 − ϕ) depending on how it affects the TSE. High ϕ-values (>0.7) indicate a TSE as perturbed as the native state, and low ϕ-values (<0.3) little effects on the TSE. ϕ-values are viewed as probes of the degree of native structure present in the TSE at the residue level (1) with high values defining the folding nucleus (5).The large number of proteins studied experimentally with this approach has turned ϕ-values into critical...

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

“…1. For a reliable ϕ-value analysis ΔΔG 0 has to be sufficiently large (>5-7 kJ∕mol) (8,11). Table 1 shows that all thioxo-peptide bonds in the helical region except thioxo Ala4 fulfill this criterion.…”

“…This method has been successfully applied to characterize transition states for protein folding (1,2), for proteinprotein interactions (3)(4)(5) and for conformational changes in folded proteins (6). Protein folding transition states were shown to have native-like topology in the whole protein or in major parts of the structure (7)(8)(9) and it is commonly assumed that this includes the presence of native-like secondary structure (10,11). Because site-directed mutagenesis can only modify amino acid side chains, it is still unclear at which stage of a folding reaction backbone hydrogen bonds in secondary structure elements are formed.…”

Testing the diffusing boundary model for the helix–coil transition in peptides

“…S5). ϕ f -Values of about 0.3 are also found for the effect of most mutations on folding of small, single-domain proteins when only reliable ϕ f -values are considered (36,40). 5.…”

Structure of the transition state for the binding of c-Myb and KIX highlights an unexpected order for a disordered system

“…Comparative analysis of nearly all Φ-values reported to date revealed that the α-value in protein folding is robust and, for nearly all proteins investigated, α is about 0.36 (30). The mutational work reported in Table 1 is an opportunity to apply the LFER to the recognition reaction between an IDP and its physiological partner (Fig.…”