Interactions Between Small Heat Shock Proteins and Substrate Proteins Mass Spectrometric Detection of Crosslinked Peptides to Map Protein-Protein Interactions

Sammanfattning: The small heat shock proteins (sHsps) are a widespread class of molecular chaperones existing in all organisms. During heat stress the sHsps protect other proteins from unfolding and aggregation. In plants the sHsps play an especially prominent part of the stress response. In this thesis chemical crosslinking and mass spectrometric mapping of crosslinked peptides has been used to investigate the interaction between sHsps and substrate proteins. The focus has been on Hsp21, a chloroplast-localized sHsp which increases the stress resistance of Arabidopsis thaliana plants, and a thermosensitive model substrate protein, citrate synthase (CS), using the chemical crosslinker DTSSP (3,3?-dithiobis[sulfosuccinimidylpropionate]), for covalent crosslinking between lysine residues or other primary amines. In peptide array screening, binding of sHsps occurred to a peptide located in a stem-like structure protruding from the CS dimer, where the C-terminus from one CS monomer interacts with the N-terminal part of the other monomer. Mass spectrometric mapping of crosslinked Hsp21-CS peptides further confirmed interactions between Hsp21 and lysine residues (K16, K22, K432, K437) located in this stem-like structure of CS. We propose that sHsps bind to this region, which is absent in thermostable CS forms, to stabilize the Nand C-terminae and thereby prevent CS dimer dissociation and aggregation. The crosslinked Hsp21-CS peptides indicated that the substrate-binding region in the N-terminal arm in Hsp21 interacted with the thermosensitive part of CS. Similar mass spectrometric peptide mapping was also used to show how this N-terminal arm region of a mammalian sHsp, Hsp20, is involved in transglutaminase-catalyzed crosslinking of sHsps to various substrate proteins in cellular aggregates. This was studied using a peptide probe which could be crosslinked by transglutaminase to the conserved Q31 in the Nterminal arm of mammalian sHsps. Whereas the N-terminus of Hsp21 was detected in crosslinked Hsp21-Hsp21 and Hsp21-CS peptides under conditions where the Hsp21 dodecamer was intact, the C-terminal tail and a C-terminal binding groove was detected in crosslinked Hsp21-CS peptides only under conditions where the Hsp21 dodecamer dissociated. The crosslinked Hsp21-CS peptides mapped onto several sites on the CS surface, indicating that several weak and short-lived interactions between Hsp21 and CS occur already at normal temperatures. Single particle reconstruction EM of crosslinked Hsp21 in the presence of CS indicated that the crosslinker could capture the Hsp21 dodecamers in an ?activated? conformation induced by such weak and short-lived interactions with CS.