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PROTEIN STRUCTURE REPORT

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

¹ Department of Biochemistry, Lund University, Lund, S-22100 Sweden
² School of Biological Sciences, University of Wollongong, Wollongong New South Wales 2522 Australia
³ Department of Chemistry, University of Cambridge, Cambridge CB2 1TN, United Kingdom

(RECEIVED February 19, 2007; FINAL REVISION March 28, 2007; ACCEPTED April 2, 2007)

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°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.

Keywords: chemical cross-linking; mass spectrometric peptide mapping; small heat-shock protein; protein–protein interactions; citrate synthase

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