1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms.
The effects of high dilution on the 1H Fourier transform NMR spectrum of native human insulin at pH* 8.0 and 9.3 have been examined at 500 MHz resolution. The dependence of the spectrum on concentration and comparison with the spectrum of a biologically highly potent monomeric insulin mutant (SerB9----Asp) establish that at 36 microM (pH* 9.3) or 18 microM (pH* 8) and no added buffer or salts, human insulin is monomeric. Under these conditions of dilution, ionic strength, and pH*, human insulin and the SerB9----Asp mutant exhibit nearly identical 1H NMR spectra. At higher concentrations (i.e. greater than 36 microM to 0.91 mM), native human insulin dimerizes, and this aggregation causes a change in insulin conformation. Although there are many changes in the spectrum, the TyrB26 ring H3,5 proton signals located at 6.63 ppm and the methyl signal located at 0.105 ppm (characteristics of monomeric insulin) are particularly distinct signatures of the conformation change that accompanies dimerization. Magnetization transfer experiments show that the 0.105 ppm methyl signal shifts downfield to a new position at 0.45 ppm. We conclude that the 0.105 ppm methyl signal is due to a conformation in which a Leu methyl group is centered over and in van der Waals contact with the ring of an aromatic side chain. Dimerization causes a conformation change that alters this interaction, thereby causing the downfield shift. Nuclear Overhauser studies indicate that the methyl group involved is located within a cluster of aromatic side chains and that the closest ring-methyl group interaction is with the ring of PheB24.
Roy
M
M
Department of Biochemistry, University of California, Riverside 92521.
Lee
R W
RW
Brange
J
J
Dunn
M F
MF
D001224:Aspartic Acid; D002942:Circular Dichroism; D005583:Fourier Analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000235:genetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009154:Mutation; D012694:Serine
D007328:Insulin; D046911:Macromolecular Substances; D001224:Aspartic Acid; D012694:Serine