Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via 19F NMR

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• 作者：Maria Bonaccio ; Nima Ghaderi ; Dan Borchardt ; and Michael F. Dunn
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：May 31, 2005
• 年：2005
• 卷：44
• 期：21
• 页码：7656 - 7668
• 全文大小：743K
• 年卷期：v.44,no.21(May 31, 2005)
• ISSN：1520-4995

文摘

The insulin hexamer is an allosteric protein widely used in formulations for the treatment ofdiabetes. The hexamer exhibits positive and negative cooperativity and apparent half-site binding activity,reflecting the interconversion of three allosteric states, designated as T₆, T₃R₃, and R₆. The hexamer containstwo symmetry-related Zn²⁺ located 16 Å apart on the 3-fold symmetry axis. In the transition of T₃ unitsto R₃ units, Zn²⁺ switches from an octahedral Zn²⁺N₃O₃ complex (N is HisB10, O is H₂O) to a distortedtetrahedral Zn²⁺N₃L complex (L is a monovalent anion). Hence, monovalent anions are allosteric ligandsthat stabilize R₃ units of T₃R₃ and R₆. Herein, we exploit the high sensitivity of ¹⁹F NMR chemical shiftsand fluorinated carboxylates to reveal subtle differences in the anion-binding sites of T₃R₃ and R₆. Weshow that the chemical shifts of 4- and 3-trifluoromethylbenzoate and 4- and 2-trifluoromethylcinnamategive bound resonances that distinguish between T₃R₃ and R₆. 3-Trifluoromethylbenzoate and 2-trifluoromethylcinnamate also were shown to bind to the R₃ units of T₃R₃ and R₆ in two alternative, slowlyinterconverting modes with different microenvironments for the CF₃ groups. Line width analysis showsthat ligand off rates are slower by 1/10³ than the diffusion limit, indicating a rate-limiting proteinconformational 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.