Mackenzie Harold W, Hansen D Flemming
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, WC1E 6BT, UK.
J Biomol NMR. 2017 Nov;69(3):123-132. doi: 10.1007/s10858-017-0137-2. Epub 2017 Nov 10.
Arginine side-chains are often key for enzyme catalysis, protein-ligand and protein-protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine C-detected NMR experiment in which a double-quantum coherence involving the two N nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two N; this new approach is shown to eliminate the previously deleterious line broadenings of N resonances caused by the partially restricted rotation about the C-N bond. Consequently, sharp and well-resolved N resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine N nuclei becomes possible using the double-quantum experiment.
精氨酸侧链通常是酶催化、蛋白质-配体和蛋白质-蛋白质相互作用的关键。精氨酸的重要性源于其末端胍基能够形成许多关键相互作用，如氢键和盐桥，以及其永久正电荷。我们在此展示了一种精氨酸碳检测核磁共振实验，其中涉及两个氮核的双量子相干在间接化学位移演化期间进行演化。由于双量子相干的进动频率对两个氮的交换不敏感；这种新方法被证明可以消除先前由围绕碳-氮键的部分受限旋转引起的氮共振有害线宽展。因此，可以观察到尖锐且分辨率良好的氮共振。所提出方法的实用性在19 kDa蛋白质T4溶菌酶的L99A突变体上得到了证明，在该突变体中，使用双量子实验可以测量精氨酸胺氮核的小化学位移扰动，如一键氘同位素位移。
