Combining experimental and simulation data to describe all of the structures and the pathways involved in folding a protein is problematical. Transition states can be mapped experimentally by phi values, but the denatured state is very difficult to analyse under conditions that favour folding. Also computer simulation at atomic resolution is currently limited to about a microsecond or less. Ultrafast-folding proteins fold and unfold on timescales accessible by both approaches, so here we study the folding pathway of the three-helix bundle protein Engrailed homeodomain. Experimentally, the protein collapses in a microsecond to give an intermediate with much native alpha-helical secondary structure, which is the major component of the denatured state under conditions that favour folding. A mutant protein shows this state to be compact and contain dynamic, native-like helices with unstructured side chains. In the transition state between this and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion-collision model. Molecular dynamics simulations give rate constants and structural details highly consistent with experiment, thereby completing the description of folding at atomic resolution.

This review describes how kinetic experiments using techniques with dramatically improved time resolution have contributed to understanding mechanisms in protein folding. Optical triggering with nanosecond laser pulses has made it possible to study the fastest-folding proteins as well as fundamental processes in folding for the first time. These include formation of alpha-helices, beta-sheets, and contacts between residues distant in sequence, as well as overall collapse of the polypeptide chain. Improvements in the time resolution of mixing experiments and the use of dynamic nuclear magnetic resonance methods have also allowed kinetic studies of proteins that fold too fast (greater than approximately 10(3) s-1) to be observed by conventional methods. Simple statistical mechanical models have been extremely useful in interpreting the experimental results. One of the surprises is that models originally developed for explaining the fast kinetics of secondary structure formation in isolated peptides are also successful in calculating folding rates of single domain proteins from their native three-dimensional structure.

We have measured the kinetics of the helix-coil transition for the synthetic 21-residue peptide Ac-WAAAH+(AAAR+A)3A-NH2 initiated by nanosecond laser temperature jumps. This peptide was designed with tryptophan in position 1 and histidine in position 5 so that the side chains interact when the backbone of residues 1−5 is α-helical. Histidine, when protonated, efficiently quenches tryptophan fluorescence providing a probe for the presence of helical structure. The kinetics measured throughout the melting transition are well-described by a single-exponential relaxation, with a rate of 3.3 × 106 s-1 at 301 K, the midpoint of the helix−coil transition. The rate increases with increasing temperature with an apparent activation energy of approximately 8 kcal/mol. To interpret these results we have fitted the equilibrium and kinetic data with the statistical mechanical model of Muñoz et al. (Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5872−5879). This model includes both variable helix propensities and side chain−side chain interactions. The model accounts for the single-exponential kinetics by predicting that approximately 90% of the change in the tryptophan fluorescence results from melting of stretches of helix which include residues 1−5 by passage over a nucleation free energy barrier. The measured temperature dependence is reproduced by introducing damping from solvent friction and an activation barrier for the individual helix propagation and melting steps. This barrier is somewhat larger than that which results from the loss in conformational entropy or breaking of hydrogen bonds. The model provides a description of the kinetics of the helix-coil transition which is consistent with the results of other experimental studies as well as molecular dynamics simulations.