Folding stability modulation of the villin headpiece helical subdomain by 4‐fluorophenylalanine and 4‐methylphenylalanine

Abstract

HP36, the helical subdomain of villin headpiece, contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, and Phe58). Hydrophobic effects and electrostatic interactions were shown to be the critical factors in stabilizing this core and the global structure. To assess the interactions among Phe47, Phe51, and Phe58 residues and investigate how they affect the folding stability, we implanted 4-fluorophenylalanine (Z) and 4-methylphenylalanine (X) into the hydrophobic core of HP36. We chemically synthesized HP36 and its seven variants including four single mutants whose Phe51 or Phe58 was replaced with Z or X, and three double mutants whose Phe51 and Phe58 were both substituted. Circular dichroism and nuclear magnetic resonance measurements show that the variants exhibit a native HP36 like fold, of which F51Z and three double mutants are more stable than the wild type. Molecular modeling provided detailed interaction energy within the phenylalanine residues, revealing that electrostatic interactions dominate the stability modulation upon the introduction of 4-fluorophenylalanine and 4-methylphenylalanine. Our results show that these two non-natural amino acids can successfully tune the interactions in a relatively compact hydrophobic core and the folding stability without inducing dramatic steric effects. Such an approach may be applied to other folded motifs or proteins.

Conformational landscape of substituted prolines

“…Substitution of the Hγ at Pro62 possibly weakens the proline-aromatic C-H … π interaction, which is crucial in maintaining the local structure. Only flp (which promotes the endo ring pucker) at position 62 marginally increased the stability of the villin headpiece protein, which is attributed to its hydrophobic effect (Hsu et al 2015). The HPK1-derived proline rich sequence (PPPLPPKPKF) that recognizes and binds to the SH3 domain of the murine cortactin (SH3 m-cort ) via formation of the left-handed polyproline structure (Feng et al 1994;Rubini et al 2010) did not show a gain in function when exo ring pucker promoting, and P II stabilizing 4substituted prolines were incorporated in place of the proline residues (Borgogno and Ruzza 2013).…”