Analysis of molecular interactions in heat-induced aggregation of a non-inhibitory serpin ovalbumin using a molecular chaperone.
Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a Km of 165 microM and a Vmax of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a Kd of 0.52 microM by BiP, lower than that for RCMLA (Kd = 1.1 microM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (Kd = 21 microM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.
Tani
Fumito
F
Laboratory of Food and Environmental Sciences, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Goka-sho, Uji, Kyoto 611-0011, Japan. tanifumi@kais.kyoto-u.ac.jp
Shirai
Nobuaki
N
Nakanishi
Yukiko
Y
Kitabatake
Naofumi
N
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000091342:Endoplasmic Reticulum Chaperone BiP; D004797:Enzyme-Linked Immunosorbent Assay; D006360:Heat-Shock Proteins; D006358:Hot Temperature; D018832:Molecular Chaperones / Q000737:chemistry*; D010047:Ovalbumin / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D015843:Serpins / Q000737:chemistry*; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000255:Adenosine Triphosphate; D010047:Ovalbumin