title: Biophysical optimization of a therapeutic protein by nonstandard mutagenesis: studies of an iodo-insulin derivative.
abstract: Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed Lys(B28), Pro(B29)-insulin ("lispro") as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (Tyr(B26)) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ± 0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. (1)H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-Tyr(B26) modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-Tyr(B26)-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages.
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Vijay
authors.initials: V
authors.lastname: Pandyarajan
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Nelson B
authors.initials: NB
authors.lastname: Phillips
authors.affiliation: Physiology and Biophysics.
authors.forename: Gabriela P
authors.initials: GP
authors.lastname: Cox
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Yanwu
authors.initials: Y
authors.lastname: Yang
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Whittaker
authors.affiliation: Physiology and Biophysics, Medicine, and.
authors.forename: Faramarz
authors.initials: F
authors.lastname: Ismail-Beigi
authors.affiliation: From the Departments of Biochemistry, Medicine, and Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106 michael.weiss@case.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D002942:Circular Dichroism; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D008297:Male; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D051381:Rats; D011917:Rats, Inbred Lew; D011972:Receptor, Insulin / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007328:Insulin; C008147:insulin, iodo-; D011972:Receptor, Insulin
keywords: Diabetes; Hormone; Insulin; Nonstandard Mutagenesis; Nuclear Magnetic Resonance (NMR); Protein Allostery; Protein Design; Protein Stability