Abstract
The helical subdomain of villin headpiece, HP36, is one of the smallest naturally occurring cooperatively folded proteins. Because of its small size, rapid folding rate and simple three-helix topology, HP36 has been a popular model for theoretical and computational study of protein folding. Many studies have suggested that there are residual secondary structures of HP36 in the unfolded state, and considered it to be the key of fast-folding. In our study, five different combinations of double cysteine mutation were carried out on the three helices of HP36, and m-xylene was used as a cross-linker to staple the α-helix structure. Far-UV CD, NMR, and fluorescence spectra indicate that all of the mutants fold into a similar structure to that of the wild-type. By denaturation experiments, stability and cooperativity of the proteins are determined. The results show that cross-linking stabilizes the proteins, while cysteine mutation destabilizes the proteins. From the decreases of cooperativity, we conclude that α3 helix and the C-terminus of α1 helix likely maintain residual secondary structures in the unfolded state, and α2 helix may play an important role in HP36 unfolding. The final structures from MD simulation under CHARMm force field are similar to that of wild-type, and the calculated interaction energy between the residues reveals that the hydrophobicity of cysteine results in the destabilization of the proteins. Remarkably, the mutant with cross-linked α3 helix demonstrates the high stability, and the result may be related to the α3-α2 cooperative stabilizing which was reported in the literature.