Activities of Monomeric Insulin Analogs at Position A8 Are Uncorrelated with Their Thermodynamic Stabilities

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 α-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, LysB28, and ProB29 (DKP)) to create a monomeric template. Five analogs were obtained by chain combination; disulfide pairing proceeded in each case with native yield. CD and 1H 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 125I-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.