We have used laser temperature jump spectroscopy to measure the folding speed of the 20-residue Trp-cage, the smallest polypeptide known to exhibit truly cooperative folding behavior. The observed folding time (4 mus at room temperature) makes this not only the smallest foldable protein, but also the fastest, with a folding speed that exceeds contact-order predictions and approaches anticipated diffusional "speed limits" for protein folding.

We studied the microsecond folding dynamics of three ␤ hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLS aa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations. P rotein-and peptide-folding events on time scales of 1-10 s are accessible to both the fastest time-resolved experiments, such as laser temperature-jump (T-jump) spectroscopy, and to advanced simulation techniques, such as distributed computing (1-6). Combining simulation and experimental techniques in studying such systems can lead to a detailed description of folding at the molecular level, along with experimental confirmation of the predicted kinetics and thermodynamics. The ␤ hairpin, a common element in protein structures, is an important test system and a potential source of insight into the folding kinetics of larger proteins. Consequently, we have seen many inquiries into the structure and folding dynamics of ␤ hairpins in recent years (7-17). Here, we have studied Trp zippers 1-3 (TZ1-TZ3), a series of unusually stable 12-residue hairpins designed by Cochran et al. (ref. 18 and Table 1).These TZs (''TrpZips'') differ only at the turn (types IIЈ, IЈ, and D-Pro-enhanced IIЈ) and form a unique hairpin conformation in which the indole side chains from opposing pairs of Trp residues interlace to form a non-hydrogen-bonded stack or zipper along the hairpin. Our objective in this work was to explore the folding process for these peptides, as observed in hundreds of folding events simulated in atomistic molecular dynamics. To test the predicted dynamics, we compared the folding rates obtained from our simulations with experimental results from laser T-jump spectroscopy by using both Trpfluorescence and IR-absorbance probes. Materials and MethodsSimulation Methodology. Our molecular dynamics simulations used software adapted from the TINKER 3.8 (J.W. Ponder, available at http:͞͞dasher.wustl.edu͞tinker) molecular-modeling package (19). We used th...

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a Reprint requests to: Kevin W. Plaxco, Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; e-mail: kwp@chem.ucsb.edu; fax: (805) 893-4120.Abbreviations: GuHCl, guanidine hydrochloride; tris, tris hydroxymethylaminoethane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine; CD, circular dichroism. Article published online ahead of print. Article and publication date are at

Because protein folding dynamics are heavily overdamped, Kramers theory predicts the rate of folding to scale inversely with the reaction friction, which is usually interpreted to mean the solvent viscosity. This does not mean, however, that the speed of folding can increase without limit as solvent viscosity decreases. We show that, in a sufficiently fast-folding protein, the folding speed approaches a finite limit at low solvent viscosity, indicating a reaction controlled by internal friction.