Mutational analysis of the N-capping box of the α-helix of chymotrypsin inhibitor 2

Abstract

The N-terminus of the helix of the chymotrypsin inhibitor 2 from barley (CI2) has an N-capping box (Ser at the first position in the helix and Glu at position 4) as well as a frequently found Glu at position 3. The energetic importance of this motif has been studied by determining the free energy of unfolding of the wild-type and protein mutants derived from those residues using guanidinium chloride-induced denaturation and differential scanning microcalorimetry. Mutating N-cap residue Ser31 to either Ala or Gly destabilizes CI2 by 0.8-1 kcal mol⁻¹. Truncation of the box in the mutants SA31EA33EA34 or SG31EA33EA34 destabilizes the protein by 1.5–2 kcal mol⁻¹. The N-capping box is an important motif in stabilizing proteins and delineating the beginning of α-helices in the pathway of protein folding.