Methods to identify the NMR resonances of the ¹³C-dimethyl N-terminal amine on reductively methylated proteins.
Methods to identify the NMR resonances of the ¹³C-dimethyl N-terminal amine on reductively methylated proteins.
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DOI：
10.3791/50875
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发表时间：
2013-12
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影响因子：
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通讯作者：
Kevin J. Roberson;Pamlea N Brady;Michelle M Sweeney;Megan A. Macnaughtan
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作者：
Kevin J. Roberson;Pamlea N Brady;Michelle M Sweeney;Megan A. Macnaughtan
核磁共振波谱是一种成熟的蛋白质结构和动态研究技术。为了用核磁共振研究蛋白质，稳定的磁性同位素通常被纳入代谢，以提高灵敏度，并允许顺序共振分配。还原（13）c -甲基化是一种替代标记方法，用于标记不适应细菌宿主过表达的蛋白质，这是最常见的同位素掺入方法。还原（13）c -甲基化是一种在温和条件下进行的化学反应，将蛋白质的初级氨基（赖氨酸ε-氨基和n端α-氨基）修饰为（13）c -二甲氨基。大多数蛋白质的结构和功能不会因修饰而改变，使其成为代谢标记的可行替代方案。由于还原（13）c -甲基化增加了稀疏的同位素标记，传统的核磁共振信号分配方法不适用。一种替代的分配方法使用质谱（MS）来帮助分配蛋白质（13）c -二甲胺核磁共振信号已经开发。该方法依赖于在每个初级氨基上部分和不同量的（13）c标记。当蛋白质的n端残基是赖氨酸时，由于Lys1的α-和ε-二甲胺基团不能用ms单独测量，因此该方法的一个局限性出现了，为了克服这一局限性，描述了两种方法来鉴定与n端α-胺和侧链ε-胺相关的（13）c -二甲胺的核磁共振。蛋清溶菌酶Lys1的n端α-二甲胺和侧链ε-二甲胺的核磁共振信号在(1)H-(13)C异核单量子相干光谱中得到了鉴定。
Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive (13)C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive (13)C-methylation is a chemical reaction performed under mild conditions that modifies a protein's primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to (13)C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive (13)C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein (13)C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of (13)C-labeling at each primary amino group. One limitation of the method arises when the protein's N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the (13)C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in (1)H-(13)C heteronuclear single-quantum coherence spectra.