Abstract
Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 +/- 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (DeltaDeltaG(u) = 0.7 +/- 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 +/- 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts Val(A3) at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 alpha-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.
Highlights
Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics
Native receptor-binding affinity (130 ؎ 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety
We first provide an overview of protein design, emphasizing both its theoretical foundation [60] and links to classical studies of insulin analogs [41,42,43,44,45, 61, 62]
Summary
Materials—Human insulin was provided by Novo Nordisk (Copenhagen, Denmark). PIP was provided by Prof. Final purification was accomplished by C18 reverse-phase high performance liquid chromatography (RP-HPLC), yielding 2.8 mg of the insulin analog. The overall yield of 2CA was similar to that obtained in control syntheses of wild-type human insulin. Redox-coupled Protein Folding—Folding of the reduced SCI-57 polypeptide was initiated by addition of a redox-coupled buffer (0.4 ml) consisting of 100 mM GSH and 10 mM GSSG; the pH was adjusted to 8.6 with 2 N NaOH This solution was immediately diluted with distilled deionized water to 40 ml. A reference structure of an engineered insulin monomer is provided by DKP-insulin (two-chain insulin analog containing three substitutions in the B-chain (AspB10, LysB28, and ProB29); Protein Data Bank code 2jmn) [7, 23]. Proteomics Tools—Molecular masses and pI values were obtained based on protein sequence using the Compute pI/Mw suite of software tools on the ExPASy Proteomics Server (ca. expasy.org/tools) with the pI algorithm (ca.expasy.org/tools/ protparam.html)
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call