Maintenance of the B-chain beta-turn in [GlyB24] insulin mutants: a steady-state fluorescence anisotropy study.

[GlyB24]insulin is a novel insulin analog which maintains nearly full biological activity [Mirmira, R. G., & Tager, H. S. (1989) J. Biol. Chem. 264, 6349-6354] even though its structure, as determined by 2D NMR, shows complete loss of the characteristic B-chain beta-turn [Hua, Q. X., Shoelson, S. E., Kochoyan, M., & Weiss, M. A. (1991) Nature 354, 238-241], which in native insulin allows the extended B-chain C-terminal region to fold against the central B-chain helix. In these studies, steady-state anisotropy measurements and fluorescence quenching analysis of the tryptophan-substituted analogs [TrpB25]insulin and [GlyB24,TrpB25]insulin have been used to study the structure of the C-terminal region of the B-chain and have demonstrated that [GlyB24]insulin mutants maintain the normal B-chain conformation to a degree comparable to that of native (PheB24) insulin at neutral pH. The tryptophan-substituted, B-chain C-terminally truncated analogs [TrpB25-alpha-carboxamide]despentapeptide(B26-B30)-insulin (DPI) and [GlyB24,TrpB25-alpha-carboxamide]DPI also significantly retain the characteristic insulin B-chain fold in solution with [GlyB24,TrpB25-alpha-carboxamide]DPI being more tightly folded than its corresponding PheB24-analog ([TrpB25-alpha-carboxamide]DPI), as assessed by these methods. The results of anisotropy measurements are consistent with the existence of a correlation between the high-affinity receptor binding of [GlyB24]insulin and the partial maintenance of the B-chain beta-turn under physiologic conditions. Thus we conclude that only analogs which possess, or can readily assume, this oriented structure can form high-affinity binding complexes with insulin receptor.