Dynamics of a Monomeric Insulin Analogue: Testing the Molten-Globule Hypothesis

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Abstract

The structure of insulin exhibits local and nonlocal differences among crystal forms and so provides an important model for analysis of protein dynamics. A novel combination of order and disorder has recently been inferred from 2D-NMR studies of the monomeric analogue des-pentapeptide(B26–B30) insulin (DPI) under acidic conditions [the molten-globule hypothesis; Hua, Q. X., Kochoyan, M., & Weiss, M. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2379–2383]. Distance-geometry structures are similar in general to crystal structures but differ by rigid-body displacements of α-helices; the hydrophobic core is not well ordered due to insufficient long-range restraints. To test whether such informational uncertainty may represent physical disorder, we have performed complementary studies of the thermal unfolding of DPI and its interaction with 1-anilino-8-naphthalenesulfonate (ANS). Experimental design is based on a predicted analogy between DPI and A-state models of protein-folding intermediates (the “molten globule”). Unfolding is monitored by five distinct biophysical probes: photochemical dynamic nuclear polarization (photo-CIDNP), differential scanning calorimetry (DSC), circular dichroism (CD), 1H-NMR chemical shifts, and slowly exchanging amide 1H-NMR resonances in D2O solution. The results provide evidence that DPI adopts a compact partially folded state. Because the 2D-NMR spectrum of an engineered insulin monomer under physiological conditions is similar to that of DPI under acidic conditions [Weiss, M. A., Hua, Q. X., Frank, B. H., Lynch, C., & Shoelson, S. E. (1991) Biochemistry 30, 7373–7389], we propose that the functional form of insulin is a molten globule. © 1993, American Chemical Society. All rights reserved.

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Hua, Q. X., Weiss, M. A., & Ladbury, J. E. (1993). Dynamics of a Monomeric Insulin Analogue: Testing the Molten-Globule Hypothesis. Biochemistry, 32(6), 1433–1442. https://doi.org/10.1021/bi00057a006

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