Liu Dongsheng, Xu Rong, Cowburn David
New York Structural Biology Center, New York, USA.
Methods Enzymol. 2009;462:151-75. doi: 10.1016/S0076-6879(09)62008-5.
Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the three-dimensional structures under near physiological conditions but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation, caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows for specific segment(s) within a protein to be selectively examined by NMR, thus significantly reducing the spectral complexity for large proteins and allowing for the application of a variety of solution-based NMR strategies. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here, we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50-kDa protein Csk (C-terminal Src kinase) using expressed protein ligation methods.
核磁共振（NMR）光谱已成为结构生物学的主要技术之一。它不仅是在接近生理条件下阐明三维结构的有力方法，也是研究蛋白质-配体相互作用和蛋白质动力学的便捷方法。大分子NMR的一个主要缺点是其尺寸限制，这是由随着尺寸增加而变慢的翻滚速率和更复杂的光谱所导致的。片段同位素标记允许通过NMR选择性地检查蛋白质内的特定片段，从而显著降低大蛋白质的光谱复杂性，并允许应用各种基于溶液的NMR策略。蛋白质的片段同位素标记通常使用两种相关方法：表达蛋白连接和蛋白质反式剪接。在此，我们描述了用于蛋白质和蛋白质复合物NMR结构研究的表达蛋白连接和蛋白质反式剪接的方法及近期应用。我们还描述了我们实验室使用表达蛋白连接方法对50 kDa的Csk（C末端Src激酶）进行片段同位素标记的方案。
