title: Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design.
abstract: Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. We demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10----Asp, ProB28----Lys, and LysB29----Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1H NMR studies of native human insulin and a series of three related analogues--(i) the singly substituted analogue [HisB10----Asp], (ii) the doubly substituted analogue [ProB28----Lys; LysB29----Pro], and (iii) DKP-insulin--demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2H and 13C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid [Hua, Q. X., & Weiss, M. A. (1991) Biochemistry 30, 5505-5515]. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues.
authors.affiliation: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: M A
authors.initials: MA
authors.lastname: Weiss
authors.forename: Q X
authors.initials: QX
authors.lastname: Hua
authors.forename: C S
authors.initials: CS
authors.lastname: Lynch
authors.forename: B H
authors.initials: BH
authors.lastname: Frank
authors.forename: S E
authors.initials: SE
authors.lastname: Shoelson
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D004252:DNA Mutational Analysis; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering; D011972:Receptor, Insulin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D007328:Insulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; C070044:DKP-insulin; D001224:Aspartic Acid; D006639:Histidine; D011392:Proline; D011972:Receptor, Insulin; D014357:Trypsin; D008239:Lysine