Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin.
The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.
Roy
M
M
Department of Biochemistry, University of California, Riverside 92521-0129.
Lee
R W
RW
Kaarsholm
N C
NC
Thøgersen
H
H
Brange
J
J
Dunn
M F
MF
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D003460:Crystallization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011487:Protein Conformation
D007328:Insulin