title: Small heat shock proteins prevent aggregation of citrate synthase and bind to the N-terminal region which is absent in thermostable forms of citrate synthase.
abstract: Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889). Strong binding of sHsps was detected to a peptide corresponding to the most N-terminal alpha-helix in CS (amino acids Leu(13) to Gln(27)). The N-terminal alpha-helices in the CS dimer intertwine with the C-terminus in the other subunit and together form a stem-like structure which is protruding from the CS dimer. This stem-like structure is absent in thermostable forms of CS from thermophilic archaebacteria like Pyrococcus furiosus and Sulfolobus solfatacarium. These data therefore suggest that thermostabilization of thermosensitive CS by sHsps is achieved by stabilization of the C- and N-terminae in the protruding thermosensitive softspot, which is absent in thermostable forms of the CS dimer.
authors.affiliation: Department of Biochemistry, Lund University, Lund, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Ahrman
authors.forename: Niklas
authors.initials: N
authors.lastname: Gustavsson
authors.forename: Claus
authors.initials: C
authors.lastname: Hultschig
authors.forename: Wilbert C
authors.initials: WC
authors.lastname: Boelens
authors.forename: Cecilia Sundby
authors.initials: CS
authors.lastname: Emanuelsson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000378:metabolism; D001665:Binding Sites; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004795:Enzyme Stability; D006360:Heat-Shock Proteins / Q000378:metabolism; D050888:Heat-Shock Proteins, Small / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D040081:Protein Array Analysis; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013552:Swine; D013696:Temperature; D038203:alpha-Crystallin B Chain / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C059215:CRYAB protein, human; C400143:HSP21 protein, Arabidopsis; D006360:Heat-Shock Proteins; D050888:Heat-Shock Proteins, Small; D038203:alpha-Crystallin B Chain; D002950:Citrate (si)-Synthase