The helix-coil transition kinetics of an ␣-helical peptide were investigated by time-resolved infrared spectroscopy coupled with laser-induced temperature-jump initiation method. Specific isotope labeling of the amide carbonyl groups with 13 C at selected residues was used to obtain site-specific information. The relaxation kinetics following a temperature jump, obtained by probing the amide I band of the peptide backbone, exhibit nonexponential behavior and are sensitive to both initial and final temperatures. These data are consistent with a conformation diffusion process on the folding energy landscape, in accord with a recent molecular dynamics simulation study.T he helix-coil transition represents the simplest scenario in protein folding (1-9), yet the details of its kinetics are not understood fully. The classical helix-coil transition theory describes the mechanism of helix formation as a sequence of events, starting from a so-called nucleation step where the first helical hydrogen bond, formed between the amide carbonyl of residue i and the amide hydrogen of residue iϩ4, is generated. The subsequent steps involve helix elongation by adding an extra hydrogen bond at either end of the preexisting helical turns. It has been argued that the nucleation process encounters the largest free energy barrier during the course of helix formation because three residues concomitantly lose their conformational entropy, whereas the propagation steps are energetically favorable because only one residue loses conformational entropy that is balanced by the energy generated from the formation of one extra hydrogen bond. Provided that the nucleation barrier is large enough (compared with those encountered by the propagation steps), a two-state scenario as well as transition-state theory remains effective to explain the dynamics of helix formation. Although recent experiments on the helix-coil transition employing laser-induced temperature-jump (T-jump) method (10-13) have shown that single exponential kinetics, which are characteristic of a two-state system, seem to be adequate to describe the transition between helix-containing and nonhelixcontaining conformations, other studies involving theory and molecular dynamics (MD) simulations have suggested that the helix-coil transition may not follow first-order kinetics (14,15). Another question that is also under debate involves the rate of the nucleation process. The T-jump data of infrared (10), fluorescence (11,12), and Raman (13), as well as results from an NMR experiment (16) all suggest that the nucleation step takes place on a submicrosecond time scale, whereas a stopped-flow CD study (17) indicates that the nucleation process may be much slower, on millisecond time scales.Recently, a new view of protein folding, based on statistical mechanical models of protein-like lattice or off-lattice polymers, has gained popularity in explaining protein folding dynamics, especially inhomogeneous folding kinetics. This new view describes protein folding as parallel, diffusion-like m...

Both turn sequence and interstrand hydrophobic side-chain-sidechain interaction have been suggested to be important determinants of ␤-hairpin stability. However, their roles in controlling the folding dynamics of ␤-hairpins have not been clearly determined. Herein, we investigated the structural stability and folding kinetics of a series of tryptophan zippers by static IR and CD spectroscopies and the IR temperature jump method. Our results support a ␤-hairpin folding mechanism wherein the rate-limiting event corresponds to the formation of the turn. We find that the logarithm of the folding rate depends linearly on the entropic change associated with the turn formation, where faster folding correlates with lower entropic cost. Moreover, a stronger turn-promoting sequence increases the stability of a ␤-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a ␤-hairpin primarily by decreasing its unfolding rate.S mall size and structural simplicity make short peptides that fold into well defined structures ideal model systems for examining factors that govern protein folding (1). Of particular interest are ␤-hairpins. With two antiparallel ␤-strands connected by a turn (or loop), the ␤-hairpin motif may be regarded as the smallest folding unit that contains tertiary contacts. Although an increasing body of evidence suggests that the ␤-hairpin can act as a folding nucleus (2-4), the mechanism by which individual ␤-hairpins fold has remained elusive. This elusiveness is partly due to the fact that so far only the folding kinetics of a few sequence-unrelated ␤-hairpins have been studied experimentally (5-9). These studies firmly demonstrated that ␤-hairpins fold on the microsecond time scale; however, the marked difference in the peptide sequence of those systems studied makes it difficult to determine explicitly the key factors that control the rate of ␤-hairpin folding.Although experimental measurements of the folding kinetics of ␤-hairpins are scarce, in the past few years a remarkable number of theoretical and computational studies have been conducted regarding the folding dynamics and energetics of a variety of ␤-hairpin systems (10-21). Results from these studies generally support the idea that the peptide sequence is an important determinant of the folding rate of ␤-hairpins. For example, the statistical model of Muñoz et al. (10) predicts that moving the hydrophobic cluster one residue closer to the turn will speed up the folding rate by 4 times, whereas the results of Thirumalai and Klimov (14,17) suggest that the turn rigidity plays a rather important role in determining the rate as well as the cooperativity of ␤-hairpin folding. Although it is still under debate whether ␤-hairpin folding begins with the formation of the turn (5, 10), interstrand hydrogen bond (16), or hydrophobic collapse (11), results from simulations generally support the idea that it involves multiple kinetic events, whereas the rate-limiting step may correspond to the assem...

Here, we describe the folding͞unfolding kinetics of ␣3D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 ؎ 1.2 s (at Ϸ50°C), indicating that a protein can fold on the 1-to 5-s time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably >1 (s) ؊1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. ␣3D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways. P roteins fold along a conformational landscape that begins with a largely disorganized, unfolded structure and proceeds to a highly compacted native state with greatly reduced conformational freedom. One model for protein folding posits a conformational search that proceeds via an initial hydrophobic collapse with further folding occurring within a restricted volume (1). Although many proteins exhibit multiple stages of folding, some small, topologically simple proteins fold without detectable intermediates, indicating that the conformational search and collapse are sometimes essentially synchronous processes, which is described by the nucleation-condensation mechanism (2). This folding process can shift to the framework model with increasing secondary structure propensities (2). The folding of small proteins by either mechanism can occur on the millisecond time scale or faster (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). De novo peptide and protein design provides an attractive approach for probing the kinetics of protein folding because it allows one to define both the sequence and its resulting interactions. Unstructured peptides have been used to determine the rate of formation of the first contact between amino acid side chains in the random coil state (15-17). Also, studies on monomeric helices revealed that the formation of isolated helices occurs with a time constant of 100-300 ns (depending on the sequence) near room temperature (18, 19) and can be characterized by a relatively rough landscape (20). In contrast, ␤-hairpin formation in a model peptide occurs in Ϸ6 s (21). Finally, the role of electrostatic complementarity has been probed by examining the second-order rate of folding of designed coiled-coil peptides (22).Here, we report folding͞unfolding studies of ␣ 3 D, a de novo designed three-helix bundle protein (23). ␣ 3 D is an excellent system for probing the maximal rate of folding in a small globular protein. It was designed to be stabilized only by the packing of hydrophobic side chains and lacks buried polar residues or structured loops that might introduce significant kinetic barriers to folding (24). This protein adopts a we...