Equilibrium Intermediates in the Denaturation of Human Insulin and Two Monomeric Insulin Analogs

Abstract

The equilibrium denaturation of human insulin in a monomer-inducing solvent and of two monomeric insulin analogs, lysB28proB29 insulin and aspB10desB28-30 insulin, was reexamined [Brems, D. N., Brown, P. L., Heckenlaible, L. A., & Frank, B. H. (1990) Biochemistry 29,9289-9293] by circular dichroism (CD) at additional wavelengths in the near-UV region. Previous denaturation studies were limited by the solubility of guanidine hydrochloride being only slightly greater than the level of denaturant required to fully unfold human insulin; therefore, only a few data points were available for construction of the post-transitional baseline. In the present study, we report the use of an unfolded mimic created by enzymatic digestion of insulin to confirm the slope of the post-transitional baseline. Evidence for equilibrium unfolding intermediates for each of these insulins was indicated by noncoincidence of the denaturation transitions as monitored by tyrosine and helical-dependent CD bands (270 and 224 nm, respectively). Additional evidence for intermediates through multiphasic denaturation transitions was obtained at a wavelength likely related to disulfide conformation, 251 nm. The results suggest that for each of the insulins, at least two intermediates are significantly populated. An unfolding model is proposed in which the conformation of the least stable intermediate is slightly unfolded only in the C-terminal segment of the B chain. A second more stable intermediate retains minimal secondary structure while containing localized structure proximal to one or more of the disulfide groups. The presence of equilibrium intermediates has important implications for the folding pathway of insulin, in pharmaceutical applications such as formulation stability, and for conformational transitions that accompany receptor binding.

Toward Understanding Insulin Fibrillation

“…These results strongly suggest that fibril nucleation requires exposure of amino acid residues, normally buried in the insulin monomer, to the surface of the insulin molecule by displacement of the B-chain C-terminal from its normal position. 26 On the basis of X-ray diffraction studies 56 and NMR structural studies 57 this C-terminal segment has been shown to be highly flexible and easily displaced, and an equilibrium unfolding intermediate involving such displacement has recently been proposed 58 (Figure 7). …”

Cross-Linking Electrochemical Mass Spectrometry for Probing Protein Three-Dimensional Structures

“…This result agrees with the solvent accessibility (shown in Table S-2, Supporting Information) that the B chain N terminus is solvent accessible for native insulin while the lysine residue K 29 is not. However, the failure of the reaction with the A chain N terminus might be due to the possibility that the N terminus of the A chain is involved in salt bridge formation 63 so that it becomes less solvent accessible.…”

In Vitro Folding/Unfolding of Insulin/Single-Chain Insulin

“…Bryant et al [26] detected two distinct transitions at 1.5 M GdnHCl and 4.5 M GdnHCl, respectively, as equilibrium intermediates in the unfolding of insulin. From the study of, Millican et al [27] based on the unfolding of human insulin and two monomeric insulin, two intermediates were obtained: one was unstable, in which only the C-terminal segment of B chain unfolded; the other was quite stable, in which little secondary structures were retained and they localized proximally to one or more of the disulfide groups.…”

“…Previous studies completed on the unfolding process of insulin or proinsulin were often carried out using denaturation method, in which disulfides remain intact [26,27]. The unfolding of insulin and proinsulin using reductive unfolding method has not been thoroughly investigated.…”