insulin--despentapeptide(b26-b30)- and 1-anilino-8-naphthalenesulfonate

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 alpha-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.

Topics: Amino Acid Sequence; Anilino Naphthalenesulfonates; Calorimetry, Differential Scanning; Circular Dichroism; Fluorescent Dyes; Insulin; Kinetics; Macromolecular Substances; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Conformation; Protein Folding; Spectrometry, Fluorescence