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首页> 外文期刊>Biochemistry >Insulin allosteric behavior: Detection, identification, and quantification of allosteric states via F-19 NMR
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Insulin allosteric behavior: Detection, identification, and quantification of allosteric states via F-19 NMR

机译：胰岛素变构行为：通过F-19 NMR对变构状态进行检测，鉴定和定量

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摘要

The insulin hexamer is an allosteric protein widely used in formulations for the treatment of diabetes. The hexamer exhibits positive and negative cooperativity and apparent half-site binding activity, reflecting the interconversion of three allosteric states, designated as T-6, T3R3, and R-6. The hexamer contains two symmetry-related Zn2+ located 16 angstrom apart on the 3-fold symmetry axis. In the transition of T-3 units to R-3 units, Zn2+ switches from an octahedral Zn2+N3O3 complex (N is HisB10, O is H2O) to a distorted tetrahedral Zn2+N3L complex (L is a monovalent anion). Hence, monovalent anions are allosteric ligands that stabilize R-3 units of T3R3 and R-6. Herein, we exploit the high sensitivity of F-19 NMR chemical shifts and fluorinated carboxylates to reveal subtle differences in the anion-binding sites of T3R3 and R-6. We show that the chemical shifts of 4- and 3-trifluoromethylbenzoate and 4- and 2-trifluoromethylcinnamate give bound resonances that distinguish between T3R3 and R-6. 3-Trifluoromethylbenzoate and 2-trifluoromethylcinnamate also were shown to bind to the R-3 units of T3R3 and R-6 in two alternative, slowly interconverting modes with different microenvironments for the CF3 groups. Line width analysis shows that ligand off rates are slower by 1/10(3) than the diffusion limit, indicating a rate-limiting protein conformational transition. These studies confirm that the Seydoux, Malhotra, and Bernhard allosteric model (Bloom, C. R., Choi, W. E., Brzovic, P. S., Ha, J. J., Huang, S. T., Kaarsholm, N. C., and Dunn, M. F. (1995). J. Mol. Biol. 245, 324-330), provides a robust description of the insulin hexamer.
机译：胰岛素六聚体是一种变构蛋白，广泛用于治疗糖尿病的制剂中。六聚体表现出正和负的协同作用以及明显的半位点结合活性，反映出三个变构态的相互转化，分别称为T-6，T3R3和R-6。六聚体包含两个对称相关的Zn2 +，它们在3倍对称轴上相距16埃。在T-3单元向R-3单元的过渡中，Zn2 +从八面体Zn2 + N3O3配合物（N为HisB10，O为H2O）转变为扭曲的四面体Zn2 + N3L配合物（L为单价阴离子）。因此，一价阴离子是稳定T3R3和R-6的R-3单元的变构配体。在这里，我们利用F-19 NMR化学位移和氟化羧酸盐的高灵敏度来揭示T3R3和R-6的阴离子结合位点的细微差别。我们表明4-和3-三氟甲基苯甲酸酯和4-和2-三氟甲基肉桂酸酯的化学位移给出了区分T3R3和R-6的结合共振。还显示了3-三氟甲基苯甲酸酯和2-三氟甲基肉桂酸酯以两种替代的，缓慢互变的方式与T3R3和R-6的R-3单元结合，CF3基团具有不同的微环境。线宽分析表明配体关闭速率比扩散极限慢1/10（3），表明存在限速蛋白构象转变。这些研究证实了Seydoux，Malhotra和Bernhard的变构模型（Bloom，CR，Choi，WE，Brzovic，PS，Ha，JJ，Huang，ST，Kaarsholm，NC和Dunn，MF（1995）。 Biol.245，324-330）提供了胰岛素六聚体的可靠描述。

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