Choi W E, Borchardt D, Kaarsholm N C, Brzovic P S, Dunn M F
Department of Biochemistry, University of California, Riverside 92521, USA.
Proteins. 1996 Dec;26(4):377-90. doi: 10.1002/(SICI)1097-0134(199612)26:4<377::AID-PROT2>3.0.CO;2-9.
The insulin hexamer is an allosteric protein exhibiting both positive and negative cooperative homotropic interactions and positive cooperative heterotropic interactions (C. R. Bloom et al., J. Mol. Biol. 245, 324-330, 1995). In this study, detailed spectroscopic analyses of the UV/Vis absorbance spectra of the Co(II)-substituted human insulin hexamer and the 1H NMR spectra of the Zn(II)-substituted hexamer have been carried out under a variety of ligation conditions to test the applicability of the sequential (KNF) and the half-site reactivity (SMB) models for allostery. Through spectral decomposition of the characteristic d-->d transitions of the octahedral Co(II)-T-state and tetrahedral Co(II)-R-state species, and analysis of the 1H NMR spectra of T- and R-state species, these studies establish the presence of preexisting T- and R-state protein conformations in the absence of ligands for the phenolic pockets. The demonstration of preexisting R-state species with unoccupied sites is incompatible with the principles upon which the KNF model is based. However, the SMB model requires preexisting T- and R-states. This feature, and the symmetry constraints of the SMB model make it appropriate for describing the allosteric properties of the insulin hexamer.
胰岛素六聚体是一种别构蛋白，表现出正协同和负协同的同促相互作用以及正协同的异促相互作用（C.R.布卢姆等人，《分子生物学杂志》245卷，324 - 330页，1995年）。在本研究中，已在多种连接条件下对钴（II）取代的人胰岛素六聚体的紫外/可见吸收光谱以及锌（II）取代的六聚体的1H NMR光谱进行了详细的光谱分析，以测试序列（KNF）模型和半位点反应性（SMB）模型用于别构作用的适用性。通过对八面体钴（II）-T态和四面体钴（II）-R态物种特征性d→d跃迁的光谱分解，以及对T态和R态物种的1H NMR光谱分析，这些研究确定了在酚口袋没有配体的情况下预先存在的T态和R态蛋白质构象。未占据位点的预先存在的R态物种的证明与KNF模型所基于的原理不相符。然而，SMB模型要求预先存在T态和R态。这一特征以及SMB模型的对称性限制使其适合于描述胰岛素六聚体的别构性质。
