Development of the reliable methods to detect the interaction of proteins and ligands using diffusion of molecules

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There is a growing need for efficient methods, capable of reliably identifying compounds in a relatively short time span with desired binding affinity. In designing NMR-based screening methods for lead compounds, it is critical to develop the technique capable of screening a large pool of new drug candidates and identifying lead compounds with high affinity towards the target proteins. an improved version of the WaterLOGSY sequence is proposed, and its efficiency of identifying a ligand bound to a protein is compared with that of STD and NOE-pumping techniques. The effectiveness of this NMR-based screening method is demonstrated using the RNase T_1-inhibitor system.Water-LOGSY experiment was improved by incorporating the double pulsed field gradient spin-echo (DPFGSE) sequence. DPFGSE sequence provides superior selective excitation without phase distortion problems that are usually associated with conventional selective excitation pulses. In the WaterLOGSY spectra, the positive signals from the inhibitors bound to RNase T_1 were clearly observed for the H8 proton of 5'-GMP and the H2 and H8 protons of 3'-AMP. These signals were not observed in the other techniques, which indicate the effectiveness of the DPFGSE-WaterLOGSY.Practical aspects of several ROESY pulse sequences have been investigated to identify bound water molecules of the non-labeled saccharide. To increase the selectivity of water resonance excitation, very weak rf strengths for the selective 180° pulses, corresponding to pulse widths of 500 ms, was used in DPFGSE. Several Cross peaks arising from bound water proton-proton of saccharide spin-spin cross relaxation are observed, and a binding water was identified by NMR.