Mechanisms of helix induction by the closed loop.

Although various factors affecting the α-helix stability have been known, they are limited to short-range interactions that occur within the helix and its flanking regions. For better understanding of protein folding coupled with disulfide bond formation, the effect of disulfide bonds on the α-helix stability must be clarified. Using a protein fragment in which two helical regions are linked by a disulfide bond, we investigated the influence of the number of residues within the loop closed by the disulfide bond on the helix stability. We modified the number of residues within the loop by inserting glycine residues in the nonhelical region of the protein fragment. Circular dichroism and nuclear magnetic resonance spectra showed that increasing the number of inserted glycine residues led to a decreased helix content, while the helical regions remained unchanged. The helical fractions of individual residues were derived from chemical shift values, and their dependences on the number of inserted glycine residues were investigated. The results of this study and previous studies support the hypothesis that the helices are nucleated in the loop and propagated to both N- and C-termini. In addition, the fact that the helical fractions decrease with the number of inserted glycine residues suggests the mechanism in which the loop reduces the number of possible conformations, thereby promoting helix nucleation within the loop.