NDLI: Methods to identify the NMR resonances of the ¹³C-dimethyl N-terminal amine on reductively methylated proteins.

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Year: 2013 Issue: 82

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Methods to identify the NMR resonances of the ¹³C-dimethyl N-terminal amine on reductively methylated proteins.

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Methods to identify the NMR resonances of the ¹³C-dimethyl N-terminal amine on reductively methylated proteins.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Roberson, Kevin J. Brady, Pamlea N. Sweeney, Michelle M. Macnaughtan, Megan A.
Description	Author Affiliation: Roberson KJ ( Department of Chemistry, Louisiana State University.)
Abstract	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 Îµ-amino groups and the N-terminal -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 - and Îµ-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 -amine and the side chain Îµ-amine. The NMR signals of the N-terminal -dimethylamine and the side chain Îµ-dimethylamine of hen egg white lysozyme, Lys1, are identified in (1)H-(13)C heteronuclear single-quantum coherence spectra.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/50875
Journal	Journal of Visualized Experiments
Issue Number	82
Language	English
Publisher	MyJove Corp.
Publisher Date	2013-12-12
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Dimethylamines Chemistry Nuclear Magnetic Resonance, Biomolecular Proteins Animals Carbon Isotopes Isotope Labeling Methylation Muramidase Research Support, N.i.h., Extramural Video-audio Media
Content Type	Text
Resource Type	Article

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