Abstract
The effect of substituting Pro25, located in the a-helical region of the cystatin A structure, with Ser has been studied. The structures of wild type and P25S cystatin A were determined by multidimensional NMR spectroscopy under comparable conditions. These two structures were virtually identical, and the a-helix between Glu15-Lys30 exists with uninterrupted continuity, with a slight bend at residue 25. In order to characterize the possible substitution effects of Pro25 with Ser on the a-helix, the chemical shifts of the amide nitrogens and protons, the generalized order parameters obtained by the analyses of the 15N-1H relaxation data, the amide proton exchange rates, and the NOE networks among the a-helical and surrounding residues were carefully compared. None of these parameters indicated any significant static or dynamic structural differences between the a-helical regions of the wild-type and P25S cystatin A proteins. We therefore conclude that our previous structure of the wild-type cystatin A, in which the a-helix exhibited a sharp kink at Pro25, must be revised. The asymmetric distribution of hydrophobic interactions between the side-chain residues of the a-helix and the rolled b-sheet surface, as revealed by NOEs, may be responsible for the slight bend of the a-helix in both variants and for the destabilized hydrogen bonding of the a-helical residues that follow Pro25/Ser25, as evidenced by increased amide exchange rates.
