[NMR paper] Insulin allosteric behavior: detection, identification, and quantification of alloste

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Insulin allosteric behavior: detection, identification, and quantification of allosteric states via 19F NMR.

Related Articles Insulin allosteric behavior: detection, identification, and quantification of allosteric states via 19F NMR.

Biochemistry. 2005 May 31;44(21):7656-68

Authors: Bonaccio M, Ghaderi N, Borchardt D, Dunn MF

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 T6, T3R3, and R6. The hexamer contains two symmetry-related Zn2+ located 16 A apart on the 3-fold symmetry axis. In the transition of T3 units to R3 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 R3 units of T3R3 and R6. Herein, we exploit the high sensitivity of 19F NMR chemical shifts and fluorinated carboxylates to reveal subtle differences in the anion-binding sites of T3R3 and R6. We show that the chemical shifts of 4- and 3-trifluoromethylbenzoate and 4- and 2-trifluoromethylcinnamate give 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, 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.

PMID: 15909980 [PubMed - indexed for MEDLINE]



Source: PubMed