Structural comparison between wild-type and P25S human cystatin A by NMR spectroscopy. Does this mutation affect the alpha-helix conformation?

J Struct Funct Genomics. 2000;1(1):26-42. doi: 10.1023/a:1011380315619.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Cystatins / chemistry*
  • Cystatins / genetics
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry


  • Cystatins
  • Recombinant Proteins

Associated data

  • PDB/1GD3
  • PDB/1GD4