单抗探针探测蛋白质折叠机制的微量热研究

Monoclonal antibodies, a powerful tool for studying structure and function of proteins owing to their ability to recognize and bind their corresponding antigens at epitope with high efficacy and specificity, have recently become an attractive approach to elucidate the mechanism of protein folding. Monoclonal antibody McAb2C9 against Staphylococcal nuclease (SNase R) and its N-terminal fragments has been used as a probe to elucidate the mechanism of folding of these antigens. The interactions of the intact enzyme SNase R and its seven peptide fragments with McAb2C9 have been studied by isothermal microcalorimetry. The ability of the shorter peptides binding to McAb2C9 is stronger than that of the longer peptides as well as the intact enzyme, indicating that the epitope is located in the sequence of residues -6-52 of the protein. The decrease in the ability of these fragments binding with the antibody is due to the decrease in the extent of the exposure of the specific epitope and the continuous conformation adjustments of the peptides through chain elongation until the biologically active protein is formed. The above provides a new thermodynamic evidence for Tsou's hypothesis of nascent peptide folding. Thermodynamics of the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature-dependence of Gibbs free energy change. A large negative heat capacity change of the binding, -156 kJ mol-1 K-1, at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. With increasing temperature from 15.0 to 37.0 oC the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 oC the spontaneous reactivation, the PDI-assisted reactivation and the intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH were all not detected. The unfolding of rabbit muscle-type creatine kinase (MM-CK) induced by guanidine hydrochloride (GuHCl) has been studied by isothermal microcalorimetry. It has been found that the decrease in the activity of MM-CK in dilute GuHCl solutions is due to a slight perturbation of the active site conformation by dilute GuHCl. The inactivation of MM-CK precedes the overall conformation change of this enzyme during denaturation by GuHCl, providing a thermodynamic evidence for the proposition that the active site of an enzyme is situated in a limited region more flexible than the enzyme molecule as a whole. The intrinsic enthalpy, Gibbs free energy, and entropy changes for formation of an intermediate state of MM-CK in the presence of moderate GuHCl concentrations at 25.0 oC have been determined to be 260 kJ mol-1, 12.2 kJ mol-1, and 830 J mol-1 K-1, respectively. The intrinsic enthalpy, Gibbs free energy, and entropy changes for formation of the unfolded state of MM-CK at 25.0 oC have been measured as 8600 kJ mol-1, 23.0 kJ mol-1, and 29 kJ mol-1 K-1, respectively. The experimental results indicate that the unfolding of MM-CK by GuHCl exhibits remarkable enthalpy-entropy compensation and the water reorganization is involved in the unfolding reaction.

单克隆抗体作为新型分子探针,因能高效专一识别抗原决定簇而成为蛋白质折叠机制研究的匾ぞ摺Ｓ梦⒘咳确ń岷瞎馄住⑸追ㄑ芯靠菇鸹粕咸亚蚓怂崦傅タ褂肴讣安煌ざ入亩蔚南嗷プ饔煤褪侗穑竦媒岷瞎讨懈髦痔卣魅攘ρШ投ρ畔?为邹氏新生肽链折叠假说提供新的实验证据，为蛋白质折叠机制和抗原-抗体相互作用探索新的研究途径。