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Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase

Åhrman, Emma LU ; Lambert, Wietske LU ; Aquilina, J. Andrew; Robinson, Carol V. and Emanuelsson, Cecilia LU (2007) In Protein Science 16(7). p.1464-1478
Abstract
The molecular mechanism whereby the small heat-shock protein ( sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase ( CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross- linker captured Hsp21 in dodecameric form, even at increased temperature ( 47 C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21... (More)
The molecular mechanism whereby the small heat-shock protein ( sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase ( CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross- linker captured Hsp21 in dodecameric form, even at increased temperature ( 47 C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
heat-shock protein, small, chemical cross-linking, mass spectrometric peptide mapping, protein-protein interactions, citrate synthase
in
Protein Science
volume
16
issue
7
pages
1464 - 1478
publisher
The Protein Society
external identifiers
  • wos:000247465400023
  • scopus:34250810346
ISSN
1469-896X
DOI
10.1110/ps.072831607
language
English
LU publication?
yes
id
658ccde0-622f-44f9-8000-f35117544dad (old id 646175)
date added to LUP
2007-12-11 13:16:11
date last changed
2017-07-02 03:38:35
@article{658ccde0-622f-44f9-8000-f35117544dad,
  abstract     = {The molecular mechanism whereby the small heat-shock protein ( sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase ( CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross- linker captured Hsp21 in dodecameric form, even at increased temperature ( 47 C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.},
  author       = {Åhrman, Emma and Lambert, Wietske and Aquilina, J. Andrew and Robinson, Carol V. and Emanuelsson, Cecilia},
  issn         = {1469-896X},
  keyword      = {heat-shock protein,small,chemical cross-linking,mass spectrometric peptide mapping,protein-protein interactions,citrate synthase},
  language     = {eng},
  number       = {7},
  pages        = {1464--1478},
  publisher    = {The Protein Society},
  series       = {Protein Science},
  title        = {Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase},
  url          = {http://dx.doi.org/10.1110/ps.072831607},
  volume       = {16},
  year         = {2007},
}