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首页> 外文期刊>Journal of Molecular Biology >Observation of Slow Dynamic Exchange Processes in Ras Protein Crystals by 31P Solid State NMR Spectroscopy.
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Observation of Slow Dynamic Exchange Processes in Ras Protein Crystals by 31P Solid State NMR Spectroscopy.

机译：通过31P固态NMR光谱观察Ras蛋白晶体中缓慢的动态交换过程。

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摘要

The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respective molecular processes are preserved upon protein crystallization, leading, in X-ray diffraction experiments, to a blurring of the electron density map and reducing the resolution of the derived structure. Nuclear magnetic resonance (NMR) is known to be an alternative method to study molecular structure and dynamics. We designed and built a probe for phosphorus solid state NMR that allows for the first time to study static properties as well as dynamic processes in single-crystals of a protein by NMR spectroscopy. The sensitivity achieved is sufficient to detect the NMR signal from individual phosphorus sites in a 0.3mm(3) size single-crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 10(15) phosphorus nuclei. The NMR spectra obtained are discussed in terms of the conformational variability of the active center of the Ras-nucleotide complex. We conclude that, in the crystal, the protein complex exists in three different conformations. Magic angle spinning (MAS) NMR spectra of a powder sample of Ras-GppNHp show a splitting of one of the phosphate resonances and thus confirm this conclusion. The MAS spectra provide, furthermore, evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal.
机译：许多蛋白质的折叠，结构和生物学功能是蛋白质分子固有的动态特性。通常，蛋白质结晶后会保留各自的分子过程，从而在X射线衍射实验中导致电子密度图的模糊化并降低衍生结构的分辨率。已知核磁共振（NMR）是研究分子结构和动力学的另一种方法。我们设计并构建了用于磷固态NMR的探针，该探针首次允许通过NMR光谱研究蛋白质单晶中的静态特性和动态过程。达到的灵敏度足以检测到与核苷酸GppNHp结合的0.3mm（3）大小的GTPase Ras单晶中单个磷位点的NMR信号，也就是大约10（15）个磷核的信号。根据Ras-核苷酸复合物活性中心的构象变异性讨论了获得的NMR光谱。我们得出结论，在晶体中，蛋白质复合物以三种不同的构象存在。 Ras-GppNHp粉末样品的魔角旋转（MAS）NMR光谱显示磷酸酯共振之一的分裂，因此证实了这一结论。此外，MAS光谱提供了Ras蛋白晶体中缓慢，依赖温度的动态交换过程的证据。

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