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NMR structural studies of human cystatin C dimers and monomers

Ekiel, I; Abrahamson, Magnus LU ; Fulton, DB; Lindahl, P; Storer, AC; Levadoux, W; Lafrance, M; Labelle, S; Pomerleau, Y and Groleau, D, et al. (1997) In Journal of Molecular Biology 271(2). p.266-277
Abstract
Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, “trapped” dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance... (More)
Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, “trapped” dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H-15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric. (Less)
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cystatin C, protein self-association, NMR spectroscopy, amyloidosis, protein folding
in
Journal of Molecular Biology
volume
271
issue
2
pages
266 - 277
publisher
Elsevier
external identifiers
  • scopus:0031571596
ISSN
1089-8638
DOI
10.1006/jmbi.1997.1150
language
English
LU publication?
yes
id
626267e2-744b-4bde-a502-18f786625a78 (old id 1112448)
date added to LUP
2008-07-22 11:39:23
date last changed
2017-07-30 04:45:55
@article{626267e2-744b-4bde-a502-18f786625a78,
  abstract     = {Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, “trapped” dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H-15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.},
  author       = {Ekiel, I and Abrahamson, Magnus and Fulton, DB and Lindahl, P and Storer, AC and Levadoux, W and Lafrance, M and Labelle, S and Pomerleau, Y and Groleau, D and LeSauteur, L and Gehring, K},
  issn         = {1089-8638},
  keyword      = {cystatin C,protein self-association,NMR spectroscopy,amyloidosis,protein folding},
  language     = {eng},
  number       = {2},
  pages        = {266--277},
  publisher    = {Elsevier},
  series       = {Journal of Molecular Biology},
  title        = {NMR structural studies of human cystatin C dimers and monomers},
  url          = {http://dx.doi.org/10.1006/jmbi.1997.1150},
  volume       = {271},
  year         = {1997},
}