Abstract
The villin headpiece subdomain, HP36, is the smallest naturally occurring protein that folds cooperatively. Its small size, rapid folding, and simple three-helix topology have made it an extremely popular system for computational studies of protein folding. The role of unfolded state structure in rapid folding is an area of active investigation, but relatively little is known about the properties of unfolded states under native conditions. A peptide fragment, HP21, which contains the first and second helices of HP36 has been shown to be a good model for structure in the unfolded state of the intact domain but a detailed description of the conformational propensities of HP21 is lacking and the balance between native and nonnative interactions is not known. A series of three-dimensional NMR experiments were performed on (13)C, (15)N-labeled HP21 to investigate in detail its conformational propensities. Analysis of (13)C(alpha), (13)C(beta), (13)CO chemical shifts, Deltadelta(13)C(alpha) - Deltadelta(13)C(beta) secondary shifts, the secondary structure propensity scores, NOEs, (15)N R(2) values and comparison of experimental chemical shifts with those of HP36 and with chemical shifts calculated using the SHIFTS and SHIFTX programs all indicate that there is significant native like structure in the HP21 ensemble, and thus by implication in the unfolded state of HP36.