Peptide models provide evidence for significant structure in the denatured state of a rapidly folding protein: the villin headpiece subdomain.

The villin headpiece subdomain is a cooperatively folded 36-residue, three-alpha-helix protein. The domain is one of the smallest naturally occurring sequences which has been shown to fold. Recent experimental studies have shown that it folds on the 10-micros time scale. Its small size, simple topology, and very rapid folding have made it an attractive target for computational studies of protein folding. We present temperature-dependent NMR studies that provide evidence for significant structure in the denatured state of the headpiece subdomain. A set of peptide fragments derived from the headpiece were also characterized in order to determine if there is a significant tendency to form a locally stabilized structure in the denatured state. Peptides corresponding to each of the three isolated helices and to the connection between the first and second helices were largely unstructured. A longer peptide fragment which contains the first and second helices shows considerable structure, as judged by NMR and CD. Concentration-dependent CD measurements and analytical ultracentrifugation experiments indicate that the structure is not due to self-association. NMR studies indicate that the structure is stabilized by tertiary interactions involving phenylalanines and Val 50. A peptide in which two of the three phenylalanines are changed to leucine is considerably less structured, confirming the importance of the phenylalanines. This work indicates that there is significant structure in the denatured state of this rapidly folding protein.