title: Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability.
abstract: 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).
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, Illinois 60637.
authors.forename: Satoe H
authors.initials: SH
authors.lastname: Nakagawa
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
authors.forename: Qing-Xin
authors.initials: QX
authors.lastname: Hua
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
authors.forename: Shi-Quan
authors.initials: SQ
authors.lastname: Hu
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Jia
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
authors.forename: Shuhua
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Pharmacology & Biological Chemistry, Mt. Sinai School of Medicine, New York, New York 10029.
authors.forename: Panayotis G
authors.initials: PG
authors.lastname: Katsoyannis
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106. Electronic address: maw21@case.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: 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
chemical_list: D007328:Insulin; D011972:Receptor, Insulin; D000409:Alanine; D005998:Glycine