Early events in protein folding explored by rapid mixing methods.

As with any complex reaction, time-resolved data are essential for elucidating the mechanism of protein folding. Even in cases where the whole process of folding occurs in a single step, which is the case for many small proteins [1], the kinetics of folding and unfolding provide valuable information on the rate-limiting barrier. The effects of temperature and denaturant concentration give insight into activation energies and solvent-accessibility of the transition state ensemble, and by measuring the kinetic effects of mutations, one can gain more detailed structural insight [2–4]. If the protein folding process occurs in stages, i.e., if partially structured intermediate states accumulate, kinetic studies can potentially offer much additional insight into the structural and thermodynamic properties of intermediate states and intervening barriers [5–9]. Rapid mixing techniques have played a prominent role in kinetic studies of protein folding [5–7, 10–12]. The combination of quenched-flow techniques with hydrogen exchange labeling and NMR has proven to be particularly fruitful for the structural characterization of transient folding intermediates [13–15]. Theoretical models and computer simulations describe the process of protein folding in terms of a diffusive motion of a particle on a high-dimensional free energy surface [16–18]. This ‘‘ landscape’’ description of protein folding predicts that a protein can choose among a large number of alternative pathways, which eventually converge toward a common free energy minimum corresponding to the native structure. In contrast, the time course of protein folding monitored by optical and other experimental probes generally shows relaxation kinetics with one or a few exponential phases, which are adequately described in terms of a simple kinetic scheme with a limited number of populated states (the chemical kinetics description). These apparently conflicting models can be consolidated if the free energy surface is divided into several regions (basins) separated by substantial free energy barriers due to unfavorable enthalpic interaction or entropic factors (conformational bottlenecks). The protein can rapidly explore conformational space within each basin corresponding to a broad ensemble of unfolded or partially folded states,