Molecular picture of folding of a small alpha/beta protein

Abstract

We characterize, at the atomic level, the mechanism and thermodynamics of folding of a small alpha/beta protein. The thermodynamically significant states of segment B1 of streptococcal protein G (GB1) are probed by using the statistical mechanical methods of importance sampling and molecular dynamics. From a thermodynamic standpoint, folding commences with overall collapse, accompanied by formation of approximately 35% of the native structure. Specific contacts form at the loci experimentally inferred to be structured early in folding kinetics studies. Our study reveals that these initially structured regions are not spatially adjacent. As folding progresses, fluid-like nonlocal native contacts form, with many contacts forming and breaking as the structure searches for the native conformation. Although the alpha-helix forms early, the beta-sheet forms concomitantly with the overall topology. Water is present in the protein core up to a late stage of folding, lubricating conformational transitions during the search process. Once 80% of the native contacts have formed, water is squeezed from the protein interior and the structure descends into the native manifold. Examination of the onset of side-chain mobility within our model indicates side-chain motion is most closely linked to the overall volume of the protein and no sharp order-disorder transition appears to occur. Exploration of models for hydrogen deuterium exchange show qualitative agreement with equilibrium measurement of hydrogen/deuterium protection factors.

Figure 1

Free energy surfaces along the reaction coordinate ρ (defined by the number of native contacts) (Inset) and as a function of ρ and R_g (the radius of gyration). Contours are drawn every 0.5 kcal/mol.

Figure 2

(A) Contact maps for the native state and the initial stage of GB1 folding. Fifty-four native contacts are shown above the diagonal of the contact map, and the subset of native contacts present in the early stage of folding are shown below the diagonal. Only contacts occurring with a high probability (P > 50%) are shown, the gray scale indicates the probability for a particular contact to be formed. (B) Representative structure of GB1 during early stages of folding.

Figure 3

Number of contacts with a given probability to be formed along the folding coordinate. Probabilities are binned every 0.2 unit.

Figure 4

Number of dihedral transitions of core side chains per 100 ps of dynamics in different regions of the reaction coordinate.

Figure 5

Fractional exposure of backbone amides under native conditions calculated by two methods as described in the text (□, model 1; ▵, model 2). Experimental values for the free energy required for transient opening in hydrogen–deuterium exchange experiments on GB1 (34) (⋄). Exchange rates could not be obtained for several protons (e.g., Ala⁴⁸) because of either fast exchange or peak overlap.