Unifying features in protein-folding mechanisms

Abstract

We compare the folding of representative members of a protein superfamily by experiment and simulation to investigate common features in folding mechanisms. The homeodomain superfamily of three-helical, single-domain proteins exhibits a spectrum of folding processes that spans the complete transition from concurrent secondary and tertiary structure formation (nucleation-condensation mechanism) to sequential secondary and tertiary formation (framework mechanism). The unifying factor in their mechanisms is that the transition state for (un)folding is expanded and very native-like, with the proportion and degree of formation of secondary and tertiary interactions varying. There is a transition, or slide, from the framework to nucleation-condensation mechanism with decreasing stability of the secondary structure. Thus, framework and nucleation-condensation are different manifestations of an underlying common mechanism.

Fig. 1.

(a) Individual and superposed structures of hTRF1, c-Myb, and En-HD, with alignment of each structure about helix I. (b) Ab initio secondary structure prediction for En-HD (○), c-Myb (×), hRAP1 (♦), and hTRF1 (□). The three helices of each protein (En-HD, residues 10-22, 28-38, and 43-55; c-Myb, residues 149-162, 166-172, and 178-189; and hTRF1, residues 8-19, 26-32, and 41-51) are marked above the graph. The helical propensities were calculated by using the agadir program (32) (www.embl-heidelberg.de/cgi/agadirwrapper.pl).

Fig. 2.

Chevron plots of wild-type En-HD (○), c-Myb (×), hRAP1 (♦), and hTRF1 (□) measured in 50 mM sodium acetate buffer and 100 mM NaCl at 25°C. The lines are the best fit for a kinetic two-state model (27).

Fig. 3.

(Upper) Chevron plot of wild-type c-Myb (•) compared with a representative secondary probe in the turn region, namely G175A mutant (○). Fits follow a simple two-state model. (Lower) Chevron plots of the pseudo-wild type of the En-HD (▪) compared with a representative secondary probe mutant in the turn, namely G39A (□). Fits are the result of a three-state model. Quantitative comparison of these homologous positions in the two proteins suggests that the turn of En-HD is fully structured in the folding transition state whereas that of c-Myb is mainly unstructured.

Fig. 4.

Brønsted plot for c-Myb (•) and En-HD (□). The line is the best linear fit to the Brønsted plot for the c-Myb mutants (r = 0.90). We noted that, for En-HD, the mutants along the line with slope 0 are involved in the stabilization of H1 (A14G) and the HII-turn-HIII motif, indicating that these secondary elements are fully formed before the docking of the transition state (see text).

Fig. 5.

(a) Representative transition-state (TS) structures for independent unfolding simulations of En-HD, c-Myb, and hTRF1 at different temperatures. The TS ensembles correspond to the following time intervals: En-HD, 373_1, 1.715-1.72 ns; 373_2, 1.98-1.985 ns; 498_1, 0.165-0.17 ns; and 498_2, 0.255-0.26 ns; c-Myb, 498_1, 0.1-0.105 ns; 498_2, 0.275-0.28 ns; 498_3, 0.315-0.32 ns; 498_4, 0.225-0.23 ns; 498_5, 0.50-0.505 ns; 498_6, 0.55-0.555 ns; and 498_7, 0.145-0.150 ns; and hTRF1, 498_1, 0.14-0.145 ns; and 498_2, 0.125-0.13 ns. The structure corresponding to the final time point for each TS ensemble is presented. (b) Structures from 498-K simulations of En-HD (498_2), c-Myb (498_1), and hTRF1 (498_1).

Fig. 6.

Simplified energy diagrams for folding of small single-domain proteins. Pure nucleation-condensation implies that secondary and tertiary structures are formed simultaneously in the absence of intermediates, as observed for hTRF1. As the propensity for forming secondary structure increases, the mechanism slides from the nucleation-condensation to the pure diffusion-collision model, as observed for En-HD.