X-RAY STRUCTURE OF INSULIN MUTANTS GLYB20GLN AND GLYB8SER-GLUB13GLN: ROLES OF GLYCINE IN THE STRUCTURAL STABILITY Zhiping Yao, Yonglin Hu, Dacheng Wang Institute of Biophysics, Chinese Academy of Sciences

Two crystal structures of insulin mutants whose wild type glycine were substitute by with side-chain residues have been determined at high resolution. With the characteristic of not having sidechain, glycine plays an important role in protein 3D structure: it usually appears at the subtle position of peptide folding. and it is often highly conservative in evolution. Thus the engineered protein of glycine substitution becomes a helpful tool to investigate the function of glycine to protein conformation. Two kinds of insulin mutant, GB20Q and GB8S-EB13Q, were obtained, whose biological activity is badly decreased. It is always glycine that appears at B8 and B20 sites of wild insulin in all mammals found so far, hence great importance comes naturally for determining the structure of glycine mutant substituted by long sidechain residues. B20Q and B8S-B13Q Human Insulin mutant are both in orthorhombic crystals with the space group P2₁2₁2₁. Reflection data was collected on synchrotron radiation with Sakabe's Weissenburg Camera System in Photon Factory, Japan. The structure were solved by Molecular Replacement method, and were well refined by X-PLOR at 1.8Å and 1.6Å resolution, respectively. The final R-factor is 0.190 and 0.183, while the bond and angle RMS deviations are 0.017Å 2.535( and 0.016Å 2.281(, respectively. Substituted residues have clear density in Fo-Fc map. No big conformation changes were found, while the dimmer similar to that in 2Zn insulin could still be formed. The main chain angles (Phi and Psi) of B8 and B20 are located in the unfavorable area of Ramachandran plots, in an unstable state with high local energy. The possible relation between the structure change caused by substitution of sidechained residue for glycine and the decrease of its biological activity will be discussed with the comparison to different kinds of wild type insulin.