Structural comparison between wild-type and P25S human cystatin A by NMR spectroscopy. Does this mutation affect the alpha-helix conformation?
The effect of substituting Pro25, located in the alpha-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 alpha-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 alpha-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 alpha-helical and surrounding residues were carefully compared. None of these parameters indicated any significant static or dynamic structural differences between the alpha-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 alpha-helix exhibited a sharp kink at Pro25, must be revised. The asymmetric distribution of hydrophobic interactions between the side-chain residues of the alpha-helix and the rolled beta-sheet surface, as revealed by NOEs, may be responsible for the slight bend of the alpha-helix in both variants and for the destabilized hydrogen bonding of the alpha-helical residues that follow Pro25/Ser25, as evidenced by increased amide exchange rates.
Shimba
N
N
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo, 192-0397 Japan.
Kariya
E
E
Tate
S
S
Kaji
H
H
Kainosho
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
D015891:Cystatins; D011994:Recombinant Proteins