Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/646175
- author
- Åhrman, Emma LU ; Lambert, Wietske LU ; Aquilina, J. Andrew ; Robinson, Carol V. and Emanuelsson, Cecilia LU
- organization
- publishing date
- 2007
- 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
- 2016-04-01 12:08:22
- date last changed
- 2022-03-05 19:27:42
@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}}, keywords = {{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}}, doi = {{10.1110/ps.072831607}}, volume = {{16}}, year = {{2007}}, }