Ludvigsen S, Andersen K V, Poulsen F M
Department of Chemistry, Carlsberg Laboratory, Copenhagen, Denmark.
J Mol Biol. 1991 Feb 20;217(4):731-6. doi: 10.1016/0022-2836(91)90529-f.
A new and simple method to measure 3JHNH alpha coupling constants of proteins by adding and subtracting traces from corresponding two-dimensional nuclear Overhauser enhanced spectroscopy and two-dimensional correlated spectroscopy cross peaks after scaling is proposed. The optimal scaling for the addition and the subtraction of the two traces is obtained by minimizing an error function. The method was proven to give accurate and precise measurements of coupling constants when tested with a series of simulated spectra. The accuracy of the method was better than 0.1 Hz for all test cases including the limiting case of J = 2.0 Hz and line-width = 11.0 Hz. The accuracy of the method was better than 0.1 Hz for all test cases including The 3JHNH alpha coupling constants were measured in two-dimensional nuclear magnetic resonance spectra of the two proteins barley serine proteinase inhibitor (CI-2) and the bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens. The experimentally measured coupling constants were used to calculate the constants in a Karplus equation to be: 3JHNH alpha = 6.7 cos2(phi-60) -1.3 cos(phi-60) +1.5. These constants are in good accordance with those obtained for basic pancreatic trypsin inhibitor (BPTI). In addition, special emphasis is given to the measurements of positive phi-angles, and to the contribution of molecular dynamics on the apparent coupling constants.
提出了一种新的简单方法，通过在缩放后对相应的二维核Overhauser增强光谱和二维相关光谱交叉峰进行迹线相加和相减来测量蛋白质的3JHNHα耦合常数。通过最小化误差函数获得两条迹线相加和相减的最佳缩放比例。用一系列模拟光谱进行测试时，该方法被证明能准确、精确地测量耦合常数。在所有测试案例中，包括J = 2.0 Hz和线宽 = 11.0 Hz的极限情况，该方法的精度均优于0.1 Hz。在大麦丝氨酸蛋白酶抑制剂（CI - 2）和解淀粉芽孢杆菌的细菌核糖核酸酶（barnase）这两种蛋白质的二维核磁共振谱中测量了3JHNHα耦合常数。实验测量的耦合常数用于计算Karplus方程中的常数，结果为：3JHNHα = 6.7 cos2(φ - 60) - 1.3 cos(φ - 60) + 1.5。这些常数与碱性胰蛋白酶抑制剂（BPTI）得到的常数非常吻合。此外，特别强调了对正φ角的测量以及分子动力学对表观耦合常数的贡献。
