title: Deciphering a molecular mechanism of neonatal diabetes mellitus by the chemical synthesis of a protein diastereomer, [D-AlaB8]human proinsulin.
abstract: Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue Gly(B8) is replaced by Ser(B8). Modular total chemical synthesis was used to prepare the wild-type [Gly(B8)]proinsulin molecule and three analogs: [D-Ala(B8)]proinsulin, [L-Ala(B8)]proinsulin, and the clinical mutant [L-Ser(B8)]proinsulin. The protein diastereomer [D-Ala(B8)]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [L-Ser(B8)]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [L-Ser(B8)]proinsulin did not fold well under the physiological conditions investigated, once folded the [L-Ser(B8)]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding.
authors.affiliation: From the Departments of Chemistry, and Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637 and.
authors.forename: Michal
authors.initials: M
authors.lastname: Avital-Shmilovici
authors.affiliation: the Departments of Biochemistry and.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Whittaker
authors.affiliation: the Departments of Biochemistry and Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
authors.affiliation: From the Departments of Chemistry, and Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637 and skent@uchicago.edu.
authors.forename: Stephen B H
authors.initials: SB
authors.lastname: Kent
mesh_terms: D000409:Alanine / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003920:Diabetes Mellitus / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D007232:Infant, Newborn, Diseases / Q000378:metabolism*; D011384:Proinsulin / Q000138:chemical synthesis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism
chemical_list: D011384:Proinsulin; D000409:Alanine
keywords: Chemical Biology; Diabetes; Insulin Receptor; Mutant; Peptide Chemical Synthesis; Proinsulin; Protein Folding