title: A cavity-forming mutation in insulin induces segmental unfolding of a surrounding alpha-helix.
abstract: To investigate the cooperativity of insulin's structure, a cavity-forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, Ile(A2)-->Ala in the A1-A8 alpha-helix, does not impair disulfide pairing between chains. In accord with past studies of cavity-forming mutations in globular proteins, a decrement was observed in thermodynamic stability (DeltaDeltaG(u) 0.4-1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated alpha-helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1-A8 segment. The analog's solution structure is otherwise similar to that of native insulin, including the B chain's supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity-forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of Ile(A2), a residue of low helical propensity, stabilizes the A1-A8 alpha-helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The Ala(A2) analog's low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1-A8 alpha-helix in receptor recognition.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Xu
authors.forename: Qing-Xin
authors.initials: QX
authors.lastname: Hua
authors.forename: Satoe H
authors.initials: SH
authors.lastname: Nakagawa
authors.forename: Wenhua
authors.initials: W
authors.lastname: Jia
authors.forename: Ying-Chi
authors.initials: YC
authors.lastname: Chu
authors.forename: Panayotis G
authors.initials: PG
authors.lastname: Katsoyannis
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D007328:Insulin / Q000737:chemistry* / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature; D013816:Thermodynamics
chemical_list: D007328:Insulin; D010455:Peptides; D019791:Guanidine; D000409:Alanine