Department of Molecular Genetics, The University of Toronto, Toronto, ON, M5S 1A8, Canada.
J Biomol NMR. 2013 Mar;55(3):239-47. doi: 10.1007/s10858-013-9705-2. Epub 2013 Jan 17.
The biological environment in which a protein performs its function is a crowded milieu containing millions of molecules that can potentially lead to a great many transient, non-specific interactions. NMR spectroscopy is especially well suited to study these weak molecular contacts. Here, non-specific interactions between the Ca(2+)-bound form of calmodulin (CaM) and non-cognate proteins in Escherichia coli lysate are explored using Ile, Leu, Val and Met methyl probes. Changes in CaM methyl chemical shifts as a function of added E. coli lysate are measured to determine a minimum 'average' dissociation constant for interactions between Ca(2+)-CaM and E. coli lysate proteins. (2)H R 2 and (13)C R 1 spin relaxation rates report on the binding reaction as well. Our results further highlight the power of methyl containing side-chains for characterizing biomolecular interactions, even in complex in-cell like environments.
蛋白质发挥功能的生物环境是一个拥挤的环境，其中包含数百万种分子，这些分子可能导致许多短暂的、非特异性的相互作用。NMR 光谱特别适合研究这些弱分子接触。在这里，使用 Ile、Leu、Val 和 Met 甲基探针研究了大肠杆菌裂解物中钙结合钙调蛋白（CaM）与非同源蛋白之间的非特异性相互作用。测量 CaM 甲基化学位移随添加的大肠杆菌裂解物的变化，以确定 Ca（2+）-CaM 与大肠杆菌裂解物蛋白之间相互作用的最小“平均”离解常数。（2）H R 2 和（13）C R 1 自旋弛豫率也报告了结合反应。我们的结果进一步强调了含甲基侧链用于表征生物分子相互作用的能力，即使在复杂的类似细胞内环境中也是如此。
