MUTATIONAL ANALYSIS OF THE N-CAPPING BOX OF THE ALPHA-HELIX OF CHYMOTRYPSIN INHIBITOR-2

The N-terminus of the helix of the chymotrypsin inhibitor 2 from barle y (CI2) has an N-capping box (Ser at the first position in the helix a nd Glu at position 4) as well as a frequently found Glu at position 3. The energetic importance of this moth has been studied by determining the free energy of unfolding of the wild-type and protein mutants der ived from those residues using guanidinium chloride-induced denaturati on and differential scanning microcalorimetry. Mutating N-cap residue Ser31 to either Ala or Gly destabilizes CI2 by 0.8-1 kcal mol(-1) Trun cation of the box in the mutants SA31EA33EA34 or SG31EA33EA34 destabil izes the protein by 1.5-2 kcal mol(-1). The N-capping box is an import ant moth in stabilizing proteins and delineating the beginning of alph a-helices in the pathway of protein folding.