AnaIysis of dynamic behavior of ribonuclease upon ligand binding using high resolution NMR

酶的内部运动受配体结合限制的观点已被接受。最近，有报道称，在一些酶，如4-巴豆酸互变异构酶，鸡和人的溶菌酶的内部运动增加配体结合。现在，它是有争议的内部运动是否增加酶或不配体结合。因此，本研究中，为了阐明酶在结合配体时增加的内部运动是否是普遍的，我们制备了^<15>N均匀标记的巴斯德毕赤酵母核糖核酸酶T1，并在有或没有3 '-GMP的情况下测量了其中氮原子的弛豫时间（T_1和T_2）以及^1H和^ N之间的NOE<15>。本文<15>用无模型分析方法计算了^ N均匀标记的核糖核酸酶T1中各残基的有序参数，包括氮原子的弛豫时间T_1和T_2以及^1H和^ N之间的NOE<15>。结果表明，核糖核酸酶T1分子中的某些残基具有较小的序参量，说明核糖核酸酶T1分子在与配体结合后，其内部运动增强。

The idea that the internal motions in enzymes were restricted upon ligand binding has been accepted. Recently, it was reported that internal motions in some enzymes such 4-oxalocrotonate tautomerase, hen and human lysozymes increased upon ligand binding. Now, it is controversial whether internal motions in enzymes increase or not upon ligand binding. Therefore, in this research, in order elucidate whether the increased internal motions in enzymes upon binding its ligand is in general or not, we prepared ^<15>N uniformly labeled ribonuclease T1 from Pichia pastoris and measured the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N in them in the presence or absence of 3'-GMP. Order parameters in every residues of ^<15>N uniformly labeled ribonuclease T1 was calculated by model free analysis, of the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N. As the results, it was elucidated that some residues in ribonuclease T1 had the smaller order parameters, indicating that the internal motions in ribonuclease T1 molecule increased upon binding its ligand.