Dynamics of a monomeric insulin analogue: testing the molten-globule hypothesis.
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.
Hua
Q X
QX
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Ladbury
J E
JE
Weiss
M A
MA
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry*; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007328:Insulin; D046911:Macromolecular Substances; C007285:insulin, despentapeptide(B26-B30)-; C027132:1-anilino-8-naphthalenesulfonate