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 A22G-B31K-B32R-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 A22G amino acid which interacts with β-turn environment resulting in high flexibility of B chain C-terminus.