Reductive methylation of lysyl side-chain amines has been a successful tool in the advancement of high resolution structural biology. The utility of this method has continuously gained ground as a protein chemical modification; first, as a tool to aid protein crystallization and later, as a probe in protein nuclear magnetic resonance (NMR) spectroscopy. As an isotope-labeling strategy for NMR studies, reductive methylation has contributed to the study of protein-protein interactions and global conformational changes. While more detailed structural studies using this labeling strategy are possible, the hurdle of assigning the NMR peaks to the corresponding reductively methylated amine hinders its use. In this review, we discuss and compare strategies used to assign the NMR peaks of reductively methylated protein-amines.

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.

No abstract

No abstract