Abstract
The first part of this thesis is about stereoelectroic effects on villin headpiece subdomain (HP36). Proline can form either a Cγ exo ring pucker or a Cγ endo ring pucker. An electron-withdrawing substituent at the 4R position of proline makes proline favor an exo ring pucker, while that at the 4S position favors an endo ring pucker due to stereoelectronic effects. Stereoelectronic effects have been shown to tune the stability of a small helical protein, the Trp-cage. In the Trp-cage, increasing the exo ring pucker propensity at C-terminus of the helix can stabilize the protein. To investigate stereoelectronic effects on the N-terminus of an α-helix, we use a 36-residue helical protein, the villin headpiece subdomain (HP36), as our protein model. A few proline derivatives including (4R,2S)-4-hydroxyproline, (4R,2S)-4-methoxyproline, and (4R,2S)-4-fluoroproline were used to replace the proline in HP36. Thermal unfolding and urea denaturation measurements by CD spectroscopy have shown that the mutation destabilized the protein. Our primary results suggest that there are not only stereoelectronic effects but also proline-aromatic interactions and steric effects affecting the structure in this region. The second part of this thesis is about establishing the PPII spectrum rule by EPR. We utilized double spin labels to get the dipole-dipole moment which can be used to calculate the distance between two spin labels. A series of polyproline peptides were designed and synthesized for this purpose. The spin-labeled peptides were successfully prepared for EPR measurements. An mathematical method was applied to calculate the distance between amino acids in PPII helices. The value of 0.31 nm between two adjacent residues is consistent with the theoretical distance for a PPII helix.