Simulation, experiment, and evolution: understanding nucleation in protein S6 folding

Abstract

In this study, we explore nucleation and the transition state ensemble of the ribosomal protein S6 using a Monte Carlo (MC) Go model in conjunction with restraints from experiment. The results are analyzed in the context of extensive experimental and evolutionary data. The roles of individual residues in the folding nucleus are identified, and the order of events in the S6 folding mechanism is explored in detail. Interpretation of our results agrees with, and extends the utility of, experiments that shift phi-values by modulating denaturant concentration and presents strong evidence for the realism of the mechanistic details in our MC Go model and the structural interpretation of experimental phi-values. We also observe plasticity in the contacts of the hydrophobic core that support the specific nucleus. For S6, which binds to RNA and protein after folding, this plasticity may result from the conformational flexibility required to achieve biological function. These results present a theoretical and conceptual picture that is relevant in understanding the mechanism of nucleation in protein folding.

Fig. 1.

Two views of S6 native (a) and representative transition state (b) structures. Nucleus residues (V6, I8, I26, L30, P60, V65, and L75) are shown explicitly. Progression along the chain is indicated by color, from blue (N terminus) to red (C terminus). Images were created by using imol.

Fig. 2.

1RIS contact matrices for the native (a), TSE (b), and difference matrix (c) (Post- and Pre-TS) with the upper left corresponding to positive values and the bottom left corresponding to negative values.

Fig. 3.

S6 TSE contact statistics. (a) The average fraction of native contacts made by each reside. Circles indicate φ-restraints. (b) The average number of nonlocal (excluding n to n ± 5) contacts made by each reside. (c) CoC in S6. Residues participating in nucleation (as determined by p_fold and m-value analysis) are indicated by an x, and residues with φ^exp ≥ 0.3 are circled, highlighting agreement between CoC and φ^exp. Additionally, CoC (along with m-values and simulation) points out to some nucleus residues with apparently low φ^exp, such as L79. CoC may also identify residues with functional importance, such as I52.

Fig. 4.

Experimental m_u (a) and m_f (b) values for wild-type and mutant S6. The m-values were determined by standard procedures from the linear regions of the chevron plots, excluding regions of downward curvature that are sometimes seen at high denaturant concentrations (11). Residues in the specific nucleus display the highest m-values, indicative of alterations of the folding trajectory and loss of residual structure in the denatured ensemble for m_u and m_f, respectively.