Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, United States.
Biochemistry. 2011 Nov 1;50(43):9225-36. doi: 10.1021/bi201287e. Epub 2011 Oct 5.
Biology relies on functional interplay of proteins in the crowded and heterogeneous environment inside cells, and functional protein interactions are often weak and transient. Thus, methods that preserve these interactions and provide information about them are needed. In-cell nuclear magnetic resonance (NMR) spectroscopy is an attractive method for studying a protein's behavior in cells because it may provide residue-level structural and dynamic information, yet several factors limit the feasibility of protein NMR spectroscopy in cells; among them, slow rotational diffusion has emerged as the most important. In this paper, we seek to elucidate the causes of the dramatically slow protein tumbling in cells and in so doing to gain insight into how the intracellular viscosity and weak, transient interactions modulate protein mobility. To address these questions, we characterized the rotational diffusion of three model globular proteins in Escherichia coli cells using two-dimensional heteronuclear NMR spectroscopy. These proteins have a similar molecular size and globular fold but very different surface properties, and indeed, they show very different rotational diffusion in the E. coli intracellular environment. Our data are consistent with an intracellular viscosity approximately 8 times that of water, too low to be a limiting factor for observation of small globular proteins by in-cell NMR spectroscopy. Thus, we conclude that transient interactions with cytoplasmic components significantly and differentially affect the mobility of proteins and therefore their NMR detectability. Moreover, we suggest that an intricate interplay of total protein charge and hydrophobic interactions plays a key role in regulating these weak intermolecular interactions in cells.
生物学依赖于蛋白质在细胞内拥挤和不均匀环境中的功能相互作用，而功能蛋白相互作用通常较弱且短暂。因此，需要能够保留这些相互作用并提供相关信息的方法。细胞内核磁共振（NMR）光谱学是一种研究蛋白质在细胞内行为的有吸引力的方法，因为它可以提供残基水平的结构和动态信息，但有几个因素限制了蛋白质 NMR 光谱学在细胞中的可行性；其中，缓慢的旋转扩散已成为最重要的因素。在本文中，我们试图阐明导致蛋白质在细胞中旋转扩散明显变慢的原因，并由此深入了解细胞内粘度和弱的、短暂的相互作用如何调节蛋白质的流动性。为了解决这些问题，我们使用二维异核 NMR 光谱法对三种模型球状蛋白在大肠杆菌细胞中的旋转扩散进行了表征。这些蛋白质具有相似的分子大小和球状折叠，但表面性质却非常不同，实际上，它们在大肠杆菌细胞内环境中的旋转扩散差异很大。我们的数据与细胞内粘度约为水的 8 倍一致，这一数值太低，不足以成为细胞内 NMR 光谱法观察小球状蛋白质的限制因素。因此，我们得出结论，与细胞质成分的短暂相互作用显著且不同地影响蛋白质的流动性，从而影响它们的 NMR 可检测性。此外，我们还提出，总蛋白质电荷和疏水相互作用的复杂相互作用在调节细胞中这些弱的分子间相互作用方面起着关键作用。
