To examine whether helix formation necessarily precedes chain collision, we have measured the folding of a fully helical coiled coil that has been specially engineered to have negligible intrinsic helical propensity but high overall stability. The folding rate approaches the diffusion-limited value and is much faster than possible if folding is contingent on precollision helix formation. Therefore, the collision of two unstructured chains is the initial step of the dominant kinetic pathway, whereas helicity exerts its influence only at a later step. Folding from an unstructured encounter complex may be efficient and robust, which has implications for any biological process that couples folding to binding.ne of the most debated issues in protein folding concerns the earliest folding events leading up to the transition state (1-9). For helical proteins, the earliest productive folding steps often are postulated to involve the collision of two preformed, but not necessarily stable, helical elements, rather than collision of unstructured chains. This diffusion-collision model (D-C model) (10-12) is supported by the observation that helix formation is faster than overall folding rates (13-15). This broadly accepted view also is supported by the presence of helix in the folding transition state and an increase in k f with an increase in helical propensity (2, 4, 16-21).However, this correlation can support an opposing model in which unstructured chains first collide, and the enhanced helicity increases the success frequency (or transmission coefficient) of each encounter (Fig. 1). In general, the highly cooperative (two-state) folding behavior of most small proteins precludes identifying the order of events leading up to the kinetic barrier. As a result, the demonstration of helical structure in the transition state cannot by itself resolve whether helix formation or chain collision occurs first.This obstacle can be overcome by studying a system with minimal helical propensity and composed of more than one chain, so that the rate of collision can be varied (22). These properties enable comparisons between observed folding rates and the maximum rate consistent with a model where precollision helix formation is required. Our investigation uses this strategy in conjunction with a dimeric coiled coil protein specially engineered to have negligible intrinsic helicity but high stability. This protein folds at nearly the diffusion limit and certainly at a much faster rate than would be possible if the helix must form before collision. Thus, an unstructured encounter complex can successfully initiate rapid folding, with helix formation occurring at a later step. The collision-first route sets a high basal level for the folding rate of any protein.

Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to ''zipper up,'' while having an extrapolated folding rate of kf ‫؍‬ 2 ؋ 10 5 s ؊1 . These properties make it likely to attain the elusive ''downhill folding'' limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. protein folding ͉ EX1 ͉ stretched exponential ͉ coiled-coil T he two-state approximation of protein folding, in which the unfolded and native states are separated by a single freeenergy barrier and no other species accumulate to a significant degree, appears to be adequate for most small proteins (1). Nevertheless, it is unsatisfying because it generally precludes identifying the individual steps involved in the folding process. To overcome this problem, experiments (2-6) have pursued the elusive theoretical prediction of ''downhill folding'' (7-9) (Fig. 1). If attainable, a downhill energy surface may allow the experimental identification of a series of intermediate states along the folding pathways that likely exists but largely has been observed only in computer simulations.Downhill-folding behavior is predicted to occur when folding rates approach the value of the attempt frequency of the barrier crossing process,Here, the barrier height does not contribute to the rate, and ⌬G ‡ Ϸ0. This limit may be likened to extreme Hammond behavior (10) in which the folding transition state moves so far to the starting condition in response to heightened stability that the transition state coincides with the unfolded state.A barrier-free reaction must be extremely rapid, but how fast is fast enough? Estimates from measured rates of helix, loop, and hairpin formation, reaction-rate and polymer-collapse theories, and folding simulations suggest that the minimum possible folding time constant is on the order of 1 s (6). Because of the increased ruggedness of the energy landscape, downhill folders of high stability would have proportionally slower folding rates, as would those with longer sequences and greater ␤-sheet content. After correcting for length and stability, 12 proteins with predicted barrier-free time constants of Ͻ100 s have been identified as potential downhill folders (6).The folding speed and stability of the covalently cross-linked variant of the dimeric yeast transcription factor GCN4 coiledcoil places it with the top members of this group (11,12). For the version GCN4p2C, with the Gly-Gly-Cys tether located at the C terminus, the extrapolated folding time constant is f Ϸ 10 s and Ͻ1 s when normalized for the high stability of the molecule (6). This fast folding rate may be due to a simple topology that requires that the helices only ''zipper-up.'' These qualities identify it among the most likely candidates for downhill folding. Here, we invest...