Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
Biochem Soc Trans. 2010 Aug;38(4):940-6. doi: 10.1042/BST0380940.
For most biophysical techniques, characterization of protein-protein interactions is challenging; this is especially true with methods that rely on a physical phenomenon that is common to both of the interacting proteins. Thus, for example, in IR spectroscopy, the carbonyl vibration (1600-1700 cm(-1)) associated with the amide bonds from both of the interacting proteins will overlap extensively, making the interpretation of spectral changes very complicated. Isotope-edited infrared spectroscopy, where one of the interacting proteins is uniformly labelled with (13)C or (13)C,(15)N has been introduced as a solution to this problem, enabling the study of protein-protein interactions using IR spectroscopy. The large shift of the amide I band (approx. 45 cm(-1) towards lower frequency) upon (13)C labelling of one of the proteins reveals the amide I band of the unlabelled protein, enabling it to be used as a probe for monitoring conformational changes. With site-specific isotopic labelling, structural resolution at the level of individual amino acid residues can be achieved. Furthermore, the ability to record IR spectra of proteins in diverse environments means that isotope-edited IR spectroscopy can be used to structurally characterize difficult systems such as protein-protein complexes bound to membranes or large insoluble peptide/protein aggregates. In the present article, examples of application of isotope-edited IR spectroscopy for studying protein-protein interactions are provided.
对于大多数生物物理技术来说，蛋白质-蛋白质相互作用的特性描述具有挑战性；对于依赖于两种相互作用的蛋白质都共有的物理现象的方法尤其如此。例如，在红外光谱中，与两种相互作用的蛋白质中的酰胺键相关的羰基振动（1600-1700cm(-1)) 将广泛重叠，使得对光谱变化的解释非常复杂。为了解决这个问题，已经引入了一种同位素编辑的红外光谱技术，其中一种相互作用的蛋白质被均匀地用(13)C 或(13)C，(15)N 标记，从而能够使用红外光谱研究蛋白质-蛋白质相互作用。当一种蛋白质被(13)C 标记时，酰胺 I 带（约 45cm(-1)向低频移动）的大位移揭示了未标记蛋白质的酰胺 I 带，使其能够用作监测构象变化的探针。通过位点特异性同位素标记，可以达到单个氨基酸残基水平的结构分辨率。此外，能够记录蛋白质在不同环境中的红外光谱意味着，同位素编辑的红外光谱可用于对结合在膜上的蛋白质-蛋白质复合物或大的不溶性肽/蛋白质聚集体等困难系统进行结构特征描述。本文提供了使用同位素编辑的红外光谱研究蛋白质-蛋白质相互作用的应用实例。
