Biophysical Chemistry, Center for Molecular Protein Science, Lund University, SE-22100 Lund, Sweden.
J Am Chem Soc. 2009 Dec 30;131(51):18214-5. doi: 10.1021/ja908144y.
To probe internal motions in proteins on the 10(-8)-10(-5) s time scale by NMR relaxation, it is necessary to eliminate protein tumbling. Here, we examine to what extent magnetic relaxation dispersion (MRD) experiments on the water (1)H resonance report on protein motions in this time window. We also perform a critical test of two physically distinct mechanisms that have been proposed to explain and interpret (1)H MRD profiles from immobilized proteins: the exchange-mediated orientational randomization (EMOR) mechanism and the two-phase spin-fracton (2PSF) mechanism. For these purposes, we report the (1)H MRD profiles from protonated and partially deuterated ubiquitin, cross-linked by glutaraldehyde. The EMOR approach, with the crystal structure of ubiquitin as input, accounts quantitatively for the MRD data and shows that hydroxyl-bearing side chains undergo large-amplitude motions on the microsecond time scale. In contrast, the 2PSF model, which attributes (1)H relaxation to small-amplitude backbone vibrations that propagate in a low-dimensional fractal space, fails qualitatively in describing the effect of H-->D substitution. These findings appear to resolve the long-standing controversy over the molecular basis of water-(1)H relaxation in systems containing rotationally immobilized macromolecules, including biological tissue.
为了通过 NMR 弛豫探测蛋白质内部在 10(-8)-10(-5) s 时间尺度上的运动，有必要消除蛋白质的翻滚。在这里，我们研究了在水（1）H 共振上进行的磁共振弛豫分散（MRD）实验在多大程度上报告了此时间窗口内蛋白质的运动。我们还对两种不同的物理机制进行了严格的测试，这两种机制被提出以解释和解释固定蛋白质的（1）H MRD 谱：交换介导的取向随机化（EMOR）机制和两相自旋分形（2PSF）机制。为此，我们报告了质子化和部分氘代泛素的（1）H MRD 谱，它们通过戊二醛交联。EMOR 方法以泛素的晶体结构为输入，对 MRD 数据进行了定量解释，表明带有羟基的侧链在微秒时间尺度上发生了大幅度运动。相比之下，2PSF 模型将（1）H 弛豫归因于在低维分形空间中传播的小振幅骨架振动，它在描述 H->D 取代的影响方面存在定性上的失败。这些发现似乎解决了在包含旋转固定大分子的系统中，包括生物组织中，水-(1)H 弛豫的分子基础的长期争议。
