Protein side chain dynamics play a vital role in many biological processes, but differentiating mobile from rigid side chains remains a technical challenge in structural biology. Solution NMR spectroscopy is ideally suited for this but suffers from limited signal-to-noise, signal overlap, and a need for fractional ¹³C or ²H labeling. Here we introduce a simple strategy measuring initial ¹H relaxation rates during a ¹H TOCSY sequence like DIPSI-2, which can be appended to the beginning of any multi-dimensional NMR sequence that begins on ¹H. The TOCSY RF field compels all ¹H atoms to behave similarly under the influence of strong coupling and rotating frame cross-relaxation, so that differences in relaxation rates are due primarily to side chain mobility. We apply the scheme to a thermostable mutant Pin1 WW domain and demonstrate that the observed ¹H relaxation rates correlate well with two independent NMR measures of side-chain dynamics, cross-correlated ¹³C relaxation rates in ¹³C^βH₂ methylene groups and maximum observable ³J couplings sensitive to the χ₁ side chain dihedral angle (³J_Hα,Hβ, ³J_N,Hβ, and ³J_CO,Hβ). The most restricted side chains belong to Trp26 and Asn40, which are closely packed to constitute the folding center of the WW domain. None of the other conserved aromatic residues is as immobile as the first tryptophan side chain of the WW domain. The proposed ¹H relaxation methodology should make it relatively easy to measure side chain dynamics on uniformly ¹⁵N- or ¹³C-labeled proteins, so long as chemical shift assignments are obtainable.

蛋白质侧链动力学在许多生物过程中发挥着至关重要的作用，但区分移动侧链和刚性侧链仍然是结构生物学中的技术挑战。溶液 NMR 光谱非常适合此用途，但存在信噪比有限、信号重叠以及需要分数¹³ C 或² H 标记等问题。在这里，我们介绍一种简单的策略，测量^{1 H TOCSY 序列（如 DIPSI-2）期间的初始1} H 弛豫率，该策略可以附加到从¹ H开始的任何多维 NMR 序列的开头。TOCSY RF 场迫使所有¹ H原子在强耦合和旋转框架交叉弛豫的影响下表现相似，因此弛豫率的差异主要是由于侧链迁移率造成的。我们将该方案应用于热稳定突变体 Pin1 WW 结构域，并证明观察到的¹ H 弛豫率与侧链动力学的两个独立 NMR 测量、¹³ C ^β H ₂亚甲基中交叉相关的¹³ C 弛豫率和最大可观察到的³ J 耦合对 χ ₁侧链二面角敏感（³ J _Hα,Hβ、³ J _N,Hβ和³ J _CO,Hβ）。最受限制的侧链属于Trp26和Asn40，它们紧密堆积构成WW结构域的折叠中心。其他保守的芳香族残基都不像 WW 结构域的第一个色氨酸侧链那样固定。只要可以获得化学位移分配，所提出的¹ H 弛豫方法应该可以相对容易地测量统一¹⁵ N 或^{13 C 标记蛋白质的侧链动力学。}