X-Ray study on an artificial mung bean inhibitor complex with bovine β-trypsin in neat cyclohexane

摘要

The active trypsin inhibiting component, SPCl, was obtained during the synthesis of a 22-residue peptide with three disulfide bridges according to the mimic mung bean Bowman-Birk type inhibitor. The K_i value of SPCl is 1.2 * 10~(-7) M. In order to determine the topological structure of SPCl, X-ray diffraction studies were carried out on the complex of SPCl with bovine β-trypsin. Only the binding loop of SPCl resolved at 2.2 A resolution due to conformational flexibility of the other residues [1]. The amino acid sequence was re-determined and electrospray mass spectroscopy was also performed to ensure that no cleaving occurred on SPCl and the primary sequence of SPCl is correct. Because the protein is more rigid in nonaqueous medium as has been proved by others [2], we treated the complex of SPCl with neat cyclohexane and then subjected it to X-ray diffraction analysis, and the result showed that all the 22 residues of SPCl were located in the electron density map. So the topological structure of SPCl has been determined, suggesting that crystal treatment with cyclohexane may be used as a method to determine the conformation of the disordered regions in protein crystal structures.