The helix is a common secondary structural motif found in proteins, and the mechanism of helix-coil interconversion is key to understanding the protein-folding problem. We report the observation of the fast kinetics (nanosecond to millisecond) of helix melting in a small 21-residue alanine-based peptide. The unfolding reaction is initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The model peptide exhibits fast unfolding kinetics with a time constant of 160 +/- 60 ns at 28 degrees C in response to a laser-induced temperature jump of 18 degrees C which is completed within 20 ns. Using the unfolding time and the measured helix-coil equilibrium constant of the model peptide, a folding rate constant of approximately 6 x 10(7) s-1 (t1/2 = 16 ns) can be inferred for the helix formation reaction at 28 degrees C. These results demonstrate that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.

We report the fast relaxation dynamics of ''native'' apomyoglobin (pH 5.3) following a 10-ns, laserinduced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales ( ‫؍‬ 1633 cm ؊1 , ‫؍‬ 48 ns; ‫؍‬ 1650 cm ؊1 , ‫؍‬ 132 s). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm ؊1 ) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm ؊1 ). Folding rate coefficients inferred from the observed relaxations at 60؇C are k f(solvated) ‫؍‬ (7 to 20) ؋ 10 6 s ؊1 and k f(native) ‫؍‬ 3.6 ؋ 10 3 s ؊1 , respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60؇C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.The mechanisms by which a protein searches vast conformational space to attain its native fold in reasonable times and by which the three-dimensional structure is encoded in the primary sequence have not been resolved experimentally. In particular, the critical early-time structural dynamics which carry a protein along the pathway(s) from extended, disordered conformations to a compact fold are poorly characterized. A major impediment has been the conventional solutionmixing approach to initiation of a folding reaction, which imposes a short-time observation limit of greater than 1 ms.