Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability.
Insulin contains a beta-turn (residues B20-B23) interposed between two receptor-binding elements, the central alpha-helix of the B chain (B9-B19) and its C-terminal beta-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive phi dihedral angles within a classic type-I beta-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to L-amino acids, turn architecture may contribute to structure or function. Here, we employ "chiral mutagenesis," comparison of corresponding D- and L-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) phi angle. The D- and L-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the D-AlaB23 analog retains native activity, the L analog exhibits a 20-fold decrease in receptor binding. By contrast, D- and L-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (delta deltaG(u) 2.0 kcal/mole) than at B23 (delta deltaG(u) 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).
Nakagawa
Satoe H
SH
Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, Illinois 60637.
Hua
Qing-Xin
QX
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
Hu
Shi-Quan
SQ
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
Jia
Wenhua
W
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
Wang
Shuhua
S
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
Katsoyannis
Panayotis G
PG
Department of Pharmacology & Biological Chemistry, Mt. Sinai School of Medicine, New York, New York 10029.
Weiss
Michael A
MA
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106. Electronic address: maw21@case.edu.
D000409:Alanine; D017124:Conserved Sequence; D005998:Glycine; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000235:genetics*; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D011972:Receptor, Insulin / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D013816:Thermodynamics
D007328:Insulin; D011972:Receptor, Insulin; D000409:Alanine; D005998:Glycine