Abstract
Previous studies have demonstrated that the potency and thermodynamic stability of human insulin are enhanced in concert by substitution of Thr(A8) by arginine or histidine. These surface substitutions stabilize the N-terminal alpha-helix of the A chain, a key element of hormone-receptor recognition. Does enhanced stability necessarily imply enhanced activity? Here, we test by structure-based mutagenesis the relationship between the stability and activity of the hormone. To circumvent confounding effects of insulin self-association, A chain analogs were combined with a variant B chain (Asp(B10), Lys(B28), and Pro(B29) (DKP)) to create a monomeric template. Five analogs were obtained by chain combination; disulfide pairing proceeded in each case with native yield. CD and (1)H NMR spectra of the DKP analogs are essentially identical to those of DKP-insulin, indicating a correspondence of structures. Receptor binding affinities were determined by competitive displacement of (125)I-insulin from human placental membranes. Thermodynamic stabilities were measured by CD titration; unfolding was monitored as a function of guanidine concentration. In this broader collection of analogs receptor binding affinities are uncorrelated with stability. We suggest that receptor binding affinities of A8 analogs reflect local features of the hormone-receptor interface rather than the stability of the free hormone or the intrinsic C-capping propensity of the A8 side chain.
Highlights
The functional surface of insulin has long been the object of speculation (1–3)
Additional stability may derive from an electrostatic interaction between the A8 side chain and the negative charge of GluA4
To test whether the enhanced stability of HisA8 DKP-insulin is due to an electrostatic capping box involving GluA4, the latter side chain was substituted by alanine to yield the two analogs AlaA4 DKP-insulin and [AlaA4, HisA8] insulin
Summary
0.86 1.02 0.87 1.07 a Potency was measured by insulin-dependent stimulation of glucose uptake in isolated adipocytes. B Cmid is defined as that molar concentration of guanidine HCl associated with 50% protein unfolding. C The m value (kcal/mol/M) is defined as the slope in plotting the unfolding free energy versus molar concentration of denaturant; this slope is often found to be proportional to the protein surface area exposed on unfolding. D CD studies of the unfolding of human insulin represent measurements at a protein concentration of 3 M
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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 callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
