Birnbaum D T, Dodd S W, Saxberg B E, Varshavsky A D, Beals J M
Department of Biopharmaceutical Development, Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
Biochemistry. 1996 Apr 30;35(17):5366-78. doi: 10.1021/bi9600557.
Phenolic ligands, e.g., phenol and m-cresol, bind to 2Zn(II)-insulin hexamers and induce a conformational change at the N-terminus of the B-chain for each monomer. The binding of these phenolic ligands to 2Zn(II)-insulin hexamers has been studied by isothermal titrating calorimetry (ITC). The binding isotherms were modeled and thermodynamic parameters were quantified using a novel, flexible algorithm that permitted the development of a hierarchical series of physical models. With the insulin hexamer represented as a dimer of trimers, the modeling demonstrated that ligand binding is highly cooperative in nature, both intra- and inter-trimer. The isotropic inter-trimer cooperativity was dominant and negative in every system studied, with initial binding constants typically an order of magnitude greater for the binding of ligands to the first trimer relative to the second. The inter-trimer cooperatively estimated from the modeling of solution calorimetry data is consistent with a T6 <--> T3R3 <--> R6 equilibrium first proposed from crystallographic investigations. Intra-trimer cooperatively was present only in the enthalpy coefficient space, not in the equilibrium coefficient space, and therefore, less of a factor. The order of binding affinity for the ligands studied in resorcinol >> phenol > or = m-cresol as determined from their overall free energies of binding to the 2Zn(II)-insulin hexamer (-26.6, -23.4, and -23.4 kcal/mol, respectively) and their intrinsic binding constants (8780, 5040, and 3370 L/mol, respectively) at 14 degrees C. The temperature dependence of phenol binding to 2Zn(II)-insulin hexamer was modeled. Increasing temperature decreased the magnitude of both the intrinsic binding constant and the inter-trimer was cooperatively. The second phase of the ITC binding profile was also found to be highly temperature dependent. At lower temperatures the second phase is endothermic but gradually decreases with increasing temperature and subsequently becomes exothermic. This effect is attributed to loss of water from the hydration shell of the insulin hexamer with increasing temperature and consequently reduces the entropic contributions to the T <--> R transition in the phenol/2Zn(II)-insulin hexamer system.
酚类配体，如苯酚和间甲酚，可与2Zn(II)-胰岛素六聚体结合，并在每个单体的B链N端诱导构象变化。已通过等温滴定量热法（ITC）研究了这些酚类配体与2Zn(II)-胰岛素六聚体的结合。使用一种新颖、灵活的算法对结合等温线进行建模并量化热力学参数，该算法允许开发一系列分层的物理模型。将胰岛素六聚体表示为三聚体的二聚体，建模表明配体结合在本质上具有高度协同性，包括三聚体内和三聚体间。在每个研究的系统中，各向同性的三聚体间协同作用占主导且为负，相对于第二个三聚体，配体与第一个三聚体结合的初始结合常数通常大一个数量级。从溶液量热数据建模估计的三聚体间协同作用与晶体学研究首次提出的T6 <--> T3R3 <--> R6平衡一致。三聚体内协同作用仅存在于焓系数空间，而非平衡系数空间，因此影响较小。根据间苯二酚 >> 苯酚 > 或 = 间甲酚与2Zn(II)-胰岛素六聚体结合的总自由能（分别为-26.6、-23.4和-23.4 kcal/mol）及其在14℃时的固有结合常数（分别为8780、5040和3370 L/mol），确定了所研究配体的结合亲和力顺序。对苯酚与2Zn(II)-胰岛素六聚体结合的温度依赖性进行了建模。温度升高会降低固有结合常数的大小以及三聚体间的协同作用。还发现ITC结合曲线的第二阶段对温度高度敏感。在较低温度下，第二阶段是吸热的，但随着温度升高逐渐降低，随后变为放热。这种效应归因于随着温度升高，胰岛素六聚体水合壳层中的水流失，从而减少了苯酚/2Zn(II)-胰岛素六聚体系统中T <--> R转变的熵贡献。
