Pfeiffer S, Karimi-Nejad Y, Rüterjans H
Institut für Biophysikalische Chemie, Johann Wolfgang Goethe-Universität, Biozentrum, Frankfurt, Germany.
J Mol Biol. 1997 Feb 21;266(2):400-23. doi: 10.1006/jmbi.1996.0784.
Limits of NMR structure determination using multidimensional NMR spectroscopy, variable target function calculations and relaxation matrix analysis were explored using the model protein ribonuclease T1 (RNase T1). The enzyme consists of 104 amino acid residues and has a molecular mass of approximately 11 kDa. Primary experimental data comprise 1856 assigned NOE intensities, 493 3J coupling constants and 62 values of amid proton exchange rates. From these data, 2580 distance bounds, 168 allowed ranges for torsional angles and stereospecific assignments for 75% of beta-methylene protons as well as for 80% of diastereotopic methyl groups were derived. Whenever possible, the distance restraints were refined in a relaxation matrix analysis including amid proton exchange data for improvement of lower distance limits. Description of side-chain conformations were based on various models of motional averaging of 3J coupling constants. The final structure ensemble was selected from the starting ensemble comparing the global precision of structures with order parameters derived from 15N relaxation time measurements. Significant differences between the structure of RNase T1 in solution and in the crystal became apparent from a comparison of the two highly resolved structures.
使用模型蛋白核糖核酸酶T1（RNase T1）探索了利用多维核磁共振光谱、可变目标函数计算和弛豫矩阵分析进行核磁共振结构测定的局限性。该酶由104个氨基酸残基组成，分子量约为11 kDa。主要实验数据包括1856个已归属的核Overhauser效应（NOE）强度、493个3J耦合常数以及62个酰胺质子交换率值。从这些数据中，得出了2580个距离限制、168个扭转角允许范围以及75%的β-亚甲基质子和80%的非对映甲基的立体专一性归属。只要有可能，就在弛豫矩阵分析中对距离限制进行优化，该分析包括酰胺质子交换数据，以提高下限距离限制。侧链构象的描述基于3J耦合常数的各种运动平均模型。通过比较结构的整体精度与从15N弛豫时间测量得出的序参数，从起始系综中选择最终的结构系综。通过比较两个高分辨率结构，溶液中RNase T1的结构与晶体中的结构之间的显著差异变得明显。
