STRUCTURAL ASYMMETRY AND HALF-SITE REACTIVITY IN THE T TO R ALLOSTERIC TRANSITION OF THE INSULIN HEXAMER

The zinc-insulin hexamer, the storage form of insulin in the pancreas, is an allosteric protein capable of undergoing transitions between three distinct conformational states, designated T-6, T(3)R(3), and R(6), on the basis of their ligand binding properties, allosteric behavior, and pseudo point symmetries [Kaarsholm, N. C., Ko, H.-C., & Dunn, M. F. (1989) Biochemistry 28, 4427-4435]. The transition from the T-state to the R-state involves a coil-to-helix transition in residues 1-8 of the B-chain wherein the ring of PheB1 is displaced by similar to 30 Angstrom. This motion also is accompanied by small changes in the positions of A-chain residues and other B-chain residues. In this paper, one- and two-dimensional (COSY and NOESY) H-1 NMR are used to characterize the ligand-induced T to R transitions of wild-type and EB13Q mutant human zinc-insulin hexamers and to make sequence-specific assignments of all resonances in the aromatic region of the R(6) complex with resorcinol. The changes in the H-1 NMR spectrum (at 500 and 600 MHz) that occur during the T to R transition provide specific signatures of the conformation change. Analysis of the dependence of these spectral changes for the phenol-induced transition as a function of the concentration of phenol establish (1) that the interconversion of T-6 and R(6) occurs via a third species assigned as T(3)R(3) and (2) that the system shows both negative and positive cooperative allosteric behavior. One- and two-dimensional COSY and NOESY studies show that, in the absence of phenolic compounds, anions act as heterotropic effecters that shift the distribution of hexamer conformations in favor of the R-state with the order of effectiveness, SCN- > N-3(-) >> I- >> Cl-. Analysis of one- and two-dimensional spectra indicate that with wild-type insulin, SCN- and N-3(-) give T(3)R(3) species, whereas the EB13Q mutant gives an R(6) species. An allosteric model for the insulin T to R transition based on the structural asymmetry model [Seydoux, F., Malhotra, O. P., & Bernhard, S. A, (1974) CRC Crit. Rev. Biochem. 2, 227-257] is proposed that explains the negative and positive allosteric properties of the system, including the role of T(3)R(3) and the action of homotropic and heterotropic effecters.