Center for Molecular Protein Science, Department of Biophysical Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden.
J Biomol NMR. 2012 Jul;53(3):181-90. doi: 10.1007/s10858-012-9650-5. Epub 2012 Jul 3.
Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of (13)C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of samples without isotope enrichment that avoid complications due to the strong coupling between neighboring (13)C spins present in uniformly enriched proteins. Recently established protocols for specific (13)C labeling of aromatic side chains enable measurement of (13)C relaxation that can be analyzed in a straightforward manner. Here we present longitudinal- and transverse-relaxation optimized pulse sequences for measuring R (1), R (2), and {(1)H}-(13)C NOE in specifically (13)C-labeled aromatic side chains. The optimized R (1) and R (2) experiments offer an increase in sensitivity of up to 35 % for medium-sized proteins, and increasingly greater gains are expected with increasing molecular weight and higher static magnetic field strengths. Our results highlight the importance of controlling the magnetizations of water and aliphatic protons during the relaxation period in order to obtain accurate relaxation rate measurements and achieve full sensitivity enhancement. We further demonstrate that potential complications due to residual two-bond (13)C-(13)C scalar couplings or dipolar interactions with neighboring (1)H spins do not significantly affect the experiments. The approach presented here should serve as a valuable complement to methods developed for other types of protein side chains.
芳香侧链在蛋白质结合位点中很常见，它们在酶催化中发挥功能作用，并构成蛋白质疏水核心的一个组成部分。因此，研究芳香侧链的构象动力学及其对生物相关事件的响应具有重要意义。事实上，在生物分子 NMR 中，对芳香族部分（13）C 弛豫率的测量有着悠久的历史，主要是在没有同位素富集的情况下进行的，这避免了由于均匀富集的蛋白质中相邻（13）C 自旋之间的强耦合而导致的复杂性。最近建立的用于芳香侧链的特定（13）C 标记的方案可用于测量（13）C 弛豫，并且可以以直接的方式进行分析。在这里，我们提出了用于测量特定（13）C 标记的芳香侧链中的 R（1）、R（2）和{(1)H}-（13）C NOE 的纵向和横向弛豫优化脉冲序列。优化的 R（1）和 R（2）实验为中等大小的蛋白质提供了高达 35%的灵敏度提高，并且随着分子量的增加和静态磁场强度的增加，预计会有越来越大的收益。我们的结果强调了在弛豫期间控制水和脂肪族质子的磁化的重要性，以便获得准确的弛豫率测量并实现全灵敏度增强。我们进一步证明，由于与相邻（1）H 自旋的残留两键（13）C-（13）C 标量耦合或偶极相互作用引起的潜在并发症不会对实验产生显著影响。此处提出的方法应作为开发用于其他类型蛋白质侧链的方法的有价值的补充。
