Chemical Cross-linking of the Chloroplast Localized Small Heat-shock Protein, Hsp21, and the Model Substrate Citrate Synthase.
From: Department of Biochemistry, Lund University, Sweden. emma.ahrman@biochemistry.lu.se
Protein science : a publication of the Protein Society
- Publish Date: Jul 2007
- ISSN: 0961-8368
- Volume: 16
- Issue: 7
- Pages: 1464-78
- Medium: Print
- Language: English
- Citation (JAMA): Ahrman Emma, Lambert Wietske, Aquilina J Andrew, et al. Chemical Cross-linking of the Chloroplast Localized Small Heat-shock Protein, Hsp21, and the Model Substrate Citrate Synthase.. Protein Sci. Jul 2007;16:1464-78
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 3,3’-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 degrees 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.
Mesh Headings (Keywords): Amino Acid Sequence, Animals, Binding Sites, Chloroplasts, Citrate (si)-Synthase, Dimerization, Heat-Shock Proteins, Small, Mass Spectrometry, Models, Molecular, Molecular Sequence Data, Peptides, Plant Proteins, Protein Binding, Protein Denaturation, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Swine
Check for Full Text / PubMed Unique Identifier (PMID): 17567739
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