Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays.
Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.
Lentze
Nicolas
N
Institut für Mikrobiologie, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland.
Narberhaus
Franz
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D019151:Peptide Library; D038201:alpha-Crystallins; D002482:Cellulose; D002950:Citrate (si)-Synthase