Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure.
The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.
Kristensen
S M
SM
Department of Chemistry, University of Copenhagen, H.C. Orsted Institute Universitetsparken 5, Denmark.
Jørgensen
A M
AM
Led
J J
JJ
Balschmidt
P
P
Hansen
F B
FB
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D006801:Humans; D006859:Hydrogen; D007328:Insulin / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D007328:Insulin; D001224:Aspartic Acid; D006859:Hydrogen