Chemical cross‐linking of the chloroplast localized small heat‐shock protein, Hsp21, and the model substrate citrate synthase
Open Access
- 1 July 2007
- journal article
- Published by Wiley in Protein Science
- Vol. 16 (7), 1464-1478
- https://doi.org/10.1110/ps.072831607
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°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
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