Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic ¹H R_1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated ¹H–¹³C spin pairs with vicinal ²H–¹²C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the ³J_HH couplings and limited chemical shift differences among ¹H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, k_flip, agree well with previous results from ¹³C R_1ρ relaxation dispersion experiments, and yield ¹H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic¹H R_1ρ relaxation dispersion experiment in combination with the site-selective ¹H–¹³C/²H–¹²C labeling scheme enable measurement of exchange rates up to k_ex = 2k_flip = 80,000 s^–1, and serve as a useful complement to previously developed ¹³C-based methods.

芳香侧链是蛋白质动力学的有吸引力的探针，因为它们通常是酶活性位点和蛋白质结合位点的关键残基。微秒到毫秒时间尺度上的动态过程可以通过弛豫色散实验来研究，该实验通过改变以 CPMG 脉冲序列或连续自旋锁定周期实现的应用射频场的重聚焦频率来减弱构象交换对横向弛豫率的贡献。在这里，我们提出了一个芳香族¹ H R _{1 ρ}弛豫分散实验，该实验能够研究比现有芳香族侧链实验快两到三倍的交换过程。我们展示了特定位点的同位素标记方案产生孤立的由于³ J HH_耦合和^苯丙氨酸、酪氨酸^和_ _ ^_色氨酸的六环部分。这种标记模式就足够了，因为远程质子不会引起额外的并发症。我们通过测量小蛋白 GB1 中的环翻转动力学来验证该方法。确定的速率常数k _flip与¹³ C R _{1 ρ}的先前结果非常吻合弛豫色散实验，并在环两侧产生¹ H 化学位移差异，与在慢交换条件下测量的值非常吻合。芳香族¹ H R _{1 ρ}弛豫色散实验与位点选择性¹ H– ¹³ C/ ² H– ¹² C 标记方案相结合，可以测量高达k _ex  = 2 k _flip  = 80,000 s ^–1的交换率，并且作为以前开发的¹³种基于 C 的方法的有用补充。