A Mutagenesis-Free Approach to Assignment of 19F NMR Resonances in Biosynthetically Labeled Proteins
A Mutagenesis-Free Approach to Assignment of 19F NMR Resonances in Biosynthetically Labeled Proteins
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DOI：
10.1021/ja8085752
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发表时间：
2009-02-18
影响因子：
15
通讯作者：
Prosser, R. Scott
中科院分区：
文献类型：
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作者：
Kitevski-LeBlanc, Julianne L.;Al-Abdul-Wahid, M. Sameer;Prosser, R. Scott
溶液核磁共振研究蛋白质结构和动力学使用氟化氨基酸探针是一个有价值的除了现有的C-13，N-15，和H-1实验的剧目。尽管F-19核在NMR中有许多优点，但蛋白质研究因共振分配依赖于定点诱变方法而变得复杂，定点诱变方法是费力的并且经常存在问题。在这里，我们报告了一个基于核磁共振的路线分配的氟共振在C-13，N-15-3-氟-L-酪氨酸标签钙调素。分配开始与氟核的相关性的δ质子在新的C-13，N-15-富集的探针，这是使用CT-HCCF-COSY实验。通过两个额外的溶液NMR实验，即（H-B）C-B（C γ C δ）H-δ和HNCACB，进行与主链的连接。使用先前公布的骨架化学位移数据或实验获得的骨架化学位移数据完成分析，前提是在蛋白质表达期间采用统一的C-13、N-15标记程序。C-13，N-15-3-氟-L-酪氨酸探针的其他益处包括通过（（CF）-C-13-F-19）CT-HSQC减少光谱重叠，以及使用F-19 T-1、T-2和C-13-F-19 NOE监测侧链动力学的能力。
Solution NMR studies of protein structure and dynamics using fluorinated amino acid probes are a valuable addition to the repertoire of existing C-13, N-15, and H-1 experiments. Despite the numerous advantages of the F-19 nucleus in NMR, protein studies are complicated by the dependence of resonance assignments on site-directed mutagenesis methods which are laborious and often problematic. Here we report an NMR-based route to the assignment of fluorine resonances in C-13,N-15-3-fluoro-L-tyrosine tabeled calmodulin. The assignment begins with the correlation of the fluorine nucleus to the delta proton in the novel C-13, N-15-enriched probe which is achieved using a CT-HCCF-COSY experiment. Connection to the backbone is made through two additional solution NMR experiments, namely the (H-B)C-B(C gamma C delta)H-delta and HNCACB. Assignments are completed using either previously published backbone chemical shift data or obtained experimentally provided uniform C-13,N-15 labeling procedures are employed during protein expression. Additional benefits of the C-13,N-15-3-fluoro-L-tyrosine probe include the reduction of spectral overlap through ((CF)-C-13-F-19) CT-HSQCs, as welt as the ability to monitor side chain dynamics using F-19 T-1, T-2, and the C-13-F-19 NOE.