Novel recombinant insulin analogue with flexible C-terminus in B chain. NMR structure of biosynthetic engineered A22G-B31K-B32R human insulin monomer in water/acetonitrile solution

A tertiary structure of recombinant A22^G-B31^K-B32^R-human insulin monomer (insulin GKR) has been characterized by ¹H, ¹³C NMR at natural isotopic abundance using NOESY, TOCSY, ¹H/¹³C-GHSQC, and ¹H/¹³C-GHSQC-TOCSY spectra. Translational diffusion studies indicate the monomer structure in water/acetonitrile (65/35 vol.%). CSI analysis confirms existence of secondary structure motifs present in human insulin standard (HIS). Both techniques allow to establish that in this solvent recombinant insulin GKR exists as a monomer. Starting from structures calculated by the program CYANA, two different refinement protocols used molecular dynamics simulated annealing with the program AMBER; in vacuum (AMBER_VC), and including a generalized Born solvent model (AMBER_GB). From these calculations an ensemble of 20 structures of lowest energy was chosen which represents the tertiary structure of studied insulin. Here we present novel insulin with added A22^G amino acid which interacts with β-turn environment resulting in high flexibility of B chain C-terminus.