Application of Segmental Isotope Labeling Method for Protein NMR and Free Energy Calculation for Development of Inhibitors for Proteins.

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Our purpose is to build a precise structural model of a protein-ligand complex by combining the segmental isotope labeling method for protein NMR and the free energy calculation. It can be applied to development of new drugs for target proteins.As a preliminary experiment, we introduced the segmental isotope label into the maltose binding protein (MBP : 370 a.a.), and observed NMR signals. However, when the ligands were added to the protein solution, very many new resonace peaks appeared, in addition to few chemical shift changes. Thus, we concluded that MBP is not a suitable system for the current purpose. Instead, we used the b-subunit of ATP synthase, and a part of this enzyme (391-473) was labeled by ^<15>N using the segmental isotope labeling method. When the NMR signal of ^<15>N was observed, almost all the resonance peaks were identified as separated signals. By adding ADP and Mg^<2+> to this solution, we obtained the new information corresponding to the structural changes in the protein.As a consequence of investigation for 92 enzyme families for the target of drug design, D-alanyl-D-alanine peptidase (VanX : about 200 a.a.) was found to be a good candidate. We synthesized an inhibitor of VanX, and prepared the uniform labeled VaxX by ^<15>N and ^<13>C after establishing the overexpression system using E. coli. When the synthesized inhibitor was added to the VanX solution, several particular chemical shifts of the NMR peaks changed significantly, so that the active site can be identified.Simultaneously, the free energy change was estimated by docking simulation using the multicanonical WHAM upon the ligand binding to a protein. In addition, the interface between the protein and the ligand was analyzed by the saturation transfer. NMR experiment and by solving the Bloch equation during the simulated annealing. We applied this new method to the system of the CAD-ICAD complex.