National Synchrotron Light Source Seminar

The interactions which stabilize the native state and control the folding of the villin headpiece subdomain (HP36) are examined. HP36 is an extremely popular model for computational and experimental studies because of its small size, simple three-helix topology and very fast folding.
Proline-aromatic interactions involving P62 and W64 have been proposed to play a critical role in specifying the subdomain fold by acting as gatekeeper residues, i.e. as residues absolutely essential for specifying the fold. Using a stable variant of HP36 as the new background we show that proline-aromatic interactions are not required for specifying the subdomain fold. This work is important because it argues against the concept of specific gatekeeper residues.
To probe denatured state ensemble (DSE) electrostatic interactions, the pH dependent stability of wildtype HP36 and two mutants, K48M and K70M, both of which significantly increase the stability of the protein were examined. The increased stability of the K48M mutant is due to the removal of favorable electrostatic interactions in the DSE, while the increased stability of the K70M mutant is due to the introduction of a new hydrophobic interaction between the methionine and the hydrophobic core in the native state. The results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the DSE, and illustrate an approach for distinguishing native state effects from DSE effects. This work also has interesting implications for studies which attempt to stabilize proteins by targeting surface electrostatics since it shows the mechanism of stabilization may be much more complicated than anticipated.