NMR characterization of a peptide model provides evidence for significant structure in the unfolded state of the villin headpiece helical subdomain.
The villin headpiece subdomain (HP36) is the smallest naturally occurring protein that folds cooperatively. The protein folds on a microsecond time scale. Its small size and very rapid folding have made it a popular target for biophysical studies of protein folding. Temperature-dependent one-dimensional (1D) NMR studies of the full-length protein together with CD and 1D NMR studies of the 21-residue peptide fragment (HP21) derived from HP36 have shown that there is significant structure in the unfolded state of HP36 and have demonstrated that HP21 is a good model of these interactions. Here, we characterized the model peptide HP21 in detail by two-dimensional NMR. Strongly upfield shifted C(alpha) protons, the magnitude of the 3J(NH,alpha) coupling constants, and the pattern of backbone-backbone and backbone-side chain NOEs indicate that the ensemble of structures populated by HP21 contains alpha-helical structure and native as well as non-native hydrophobic contacts. The hydrogen-bonded secondary structure inferred from the NOEs is, however, not sufficient to confer significant protection against amide H-D exchange. These studies indicate that there is significant secondary structure and hydrophobic clustering in the unfolded state of HP36. The implications for the folding of HP36 are discussed.
Tang
Yuefeng
Y
Department of Chemistry, State University of New York, Stony Brook, New York 11790-3400, USA.
Goger
Michael J
MJ
Raleigh
Daniel P
DP
D000595:Amino Acid Sequence; D029721:Drosophila Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008840:Microfilament Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D029721:Drosophila Proteins; C512164:HP21 peptide; D008840:Microfilament Proteins; D016900:Neurofilament Proteins; D010446:Peptide Fragments; C026615:villin; C115223:villin headpiece subdomain peptide