Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding.

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 k(f) = 2 x 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. The stability and its denaturant dependence for the hydrogen bonds in the central part of protein equal the values calculated for whole-molecule unfolding. Like-wise, their closing and opening rates indicate that these hydrogen bonds are broken and reformed in a single cooperative event representing the folding transition from the fully unfolded state to the native state. Additionally, closing rates for these hydrogen bonds agree with the extrapolated barrier-limited folding rate observed near the melting transition. Therefore, even in the absence of denaturant, where DeltaG(eq) approximately -6 kcal.mol(-1) (1 cal = 4.18 J) and tau(f) approximately 6 mus, folding remains cooperative and barrier-limited. Given that this prime candidate for downhill folding fails to do so, we propose that protein folding will remain barrier-limited for proteins that fold cooperatively.