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

越来越需要有效的方法，能够在相对较短的时间内可靠地识别具有所需结合亲和力的化合物。在设计基于核磁共振的先导化合物筛选方法时，开发一种能够筛选大量新候选药物并识别与靶蛋白具有高亲和力的先导化合物的技术至关重要。提出了一种改进的WaterLOGSY序列，并将其识别与蛋白质结合的配体的效率与STD和noe泵送技术进行了比较。使用RNase t_1抑制剂系统证明了这种基于核磁共振的筛选方法的有效性。采用双脉冲场梯度自旋回波（DPFGSE）序列对Water-LOGSY实验进行了改进。DPFGSE序列提供了优越的选择性激励，没有通常与常规选择性激励脉冲相关的相位畸变问题。在WaterLOGSY光谱中，结合RNase T_1的抑制剂对5′-GMP的H8质子和3′-AMP的H2和H8质子有明显的正信号。这些信号在其他技术中没有观察到，这表明DPFGSE-WaterLOGSY的有效性。已经研究了几种ROESY脉冲序列的实际方面，以鉴定非标记糖的结合水分子。为了提高水共振激发的选择性，在DPFGSE中使用了选择性180°脉冲的非常弱的射频强度，对应于脉冲宽度为500 ms。观察到结合水质子-质子糖自旋-自旋交叉弛豫产生了几个交叉峰，通过核磁共振鉴定出结合水。

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.