title: Mapping the functional surface of insulin by design: structure and function of a novel A-chain analogue.
abstract: Functional surfaces of a protein are often mapped by combination of X-ray crystallography and mutagenesis. Such studies of insulin have yielded paradoxical results, suggesting that the native state is inactive and reorganizes on receptor binding. Of particular interest is the N-terminal alpha-helix of the A-chain. Does this segment function as an alpha-helix or reorganize as recently proposed in a prohormone-convertase complex? To correlate structure and function, we describe a mapping strategy based on protein design. The solution structure of an engineered monomer ([AspB10, LysB28, ProB29]-human insulin) is determined at neutral pH as a template for synthesis of a novel A-chain analogue. Designed by analogy to a protein-folding intermediate, the analogue lacks the A6-A11 disulphide bridge; the cysteine residues are replaced by serine. Its solution structure is remarkable for segmental unfolding of the N-terminal A-chain alpha-helix (A1 to A8) in an otherwise native subdomain. The structure demonstrates that the overall orientation of the A and B chains is consistent with reorganization of the A-chain's N-terminal segment. Nevertheless, the analogue's low biological activity suggests that this segment, a site of clinical mutation causing diabetes mellitus, functions as a preformed recognition alpha-helix.
authors.affiliation: Center for Molecular Oncology, University of Chicago, IL 60637, USA.
authors.forename: Q X
authors.initials: QX
authors.lastname: Hua
authors.forename: S Q
authors.initials: SQ
authors.lastname: Hu
authors.forename: B H
authors.initials: BH
authors.lastname: Frank
authors.forename: W
authors.initials: W
authors.lastname: Jia
authors.forename: Y C
authors.initials: YC
authors.lastname: Chu
authors.forename: S H
authors.initials: SH
authors.lastname: Wang
authors.forename: G T
authors.initials: GT
authors.lastname: Burke
authors.forename: P G
authors.initials: PG
authors.lastname: Katsoyannis
authors.forename: M A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017510:Protein Folding; D012996:Solutions; D013329:Structure-Activity Relationship
chemical_list: D007328:Insulin; D012996:Solutions