Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Magn Reson. 2010 Jul;205(1):114-24. doi: 10.1016/j.jmr.2010.04.011. Epub 2010 Apr 18.
Large molecules, such as membrane proteins, play crucial roles in various biologically important events. We have developed the transferred cross-saturation (TCS) method, which enables the identification of the contact residues of protein ligands in large complexes. However, rational optimization of the experimental conditions for the TCS method has been hampered by the lack of information about the influence of each experimental parameter on the observed TCS effects. Here, we established the theoretical description of the TCS method, which explicitly incorporated the isotopomers in the sample solution, and developed the computer software to perform numerical simulations. Using them, we analyzed the effects of each experimental parameter on the observed TCS effects by the simulations. The simulation studies indicated that: (i) the proton concentration in the solvent should be 10-30%, (ii) a larger pb, which is the bound fraction of the ligand, is preferred for higher saturation efficiency, (iii) the TCS method is applicable to systems where koff>0.1 s(-1), (iv) for koff>or=10 s(-1), pb>or=0.1 is preferred, (v) for koff approximately 1s(-1), pb>or=0.5 is preferred, and (vi) the TCS method is applicable to systems with large tauc (approximately 1 micros), where pb is approximately 0.01. The assumptions in the model spin simulation were experimentally verified, using the ubiquitin-YUH1 interaction. The established method will be useful for estimating and optimizing the TCS experimental conditions.
大分子量的分子，例如膜蛋白，在各种重要的生物学事件中发挥着关键作用。我们已经开发了转移交叉饱和（TCS）方法，该方法能够鉴定蛋白质配体在大复合物中的接触残基。然而，由于缺乏关于每个实验参数对观察到的 TCS 效应影响的信息，因此合理地优化 TCS 方法的实验条件一直受到阻碍。在这里，我们建立了 TCS 方法的理论描述，该描述明确地将样品溶液中的同位异构体纳入其中，并开发了用于执行数值模拟的计算机软件。使用它们，我们通过模拟分析了每个实验参数对观察到的 TCS 效应的影响。模拟研究表明：（i）溶剂中的质子浓度应为 10-30%，（ii）较大的 pb（配体的结合分数）更有利于更高的饱和效率，（iii）TCS 方法适用于 koff>0.1 s(-1)的系统，（iv）对于 koff>or=10 s(-1)，优选 pb>or=0.1，（v）对于 koff approximately 1s(-1)，优选 pb>or=0.5，以及（vi）TCS 方法适用于 tauc（大约 1 micros）较大的系统，其中 pb 大约为 0.01。使用泛素-YUH1 相互作用实验验证了模型自旋模拟中的假设。所建立的方法将有助于估计和优化 TCS 实验条件。
