School of Chemistry, NUI Galway, Ireland.
Chembiochem. 2011 May 2;12(7):1043-8. doi: 10.1002/cbic.201100063. Epub 2011 Mar 29.
Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15)N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein "stickiness" is a limiting factor in the application of in-cell NMR spectroscopy.
蛋白质科学正在向在天然或类似天然条件下进行的实验转变。例如，细胞内 NMR 光谱学有可能揭示细胞内蛋白质的结构和动态。然而，并非所有蛋白质都可以通过该技术进行研究。在大肠杆菌中过表达的 15N 标记细胞色素 c（cyt c）无法通过细胞内 NMR 光谱学检测到。当对全细胞裂解物进行大小排阻层析（SEC）时，发现 cyt c 的洗脱峰表观分子量大于 150 kDa。高分子量物质的存在表明 cyt c 与大肠杆菌胞质蛋白之间形成复合物。这些相互作用被 cyt c 的电荷反转突变体和高浓度 NaCl 破坏。生理相关的盐 KGlu 破坏复合物形成的效率较低。值得注意的是，通过 NMR 光谱学可以在细胞裂解物中检测到 cyt c 的三突变体。蛋白 GB1 产生高质量的细胞内光谱，并且含有 GB1 的裂解物的 SEC 分析显示 GB1 与大肠杆菌蛋白之间没有相互作用。这些数据表明，蛋白质“粘性”是细胞内 NMR 光谱学应用的限制因素。
