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Spectroscopic evidence for preexisting T‐ and R‐state insulin hexamer conformations
Author(s) -
Choi Wonjae E.,
Borchardt Dan,
Kaarsholm Niels C.,
Brzovic Peter S.,
Dunn Michael F.
Publication year - 1996
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(199612)26:4<377::aid-prot2>3.0.co;2-9
Subject(s) - random hexamer , allosteric regulation , chemistry , crystallography , stereochemistry , biochemistry , enzyme
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 1 H 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 1 H 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. Proteins 26:377–390 © 1996 Wiley‐Liss, Inc.