Abstract:
:13CHD2 methyl isotopomers are particularly useful to study methyl dynamics in proteins because, as compared with other methyl isotopomers, the 13C relaxation mechanism for this isotopomer is straightforward. However, in the case of proteins, where (omega tau)2 > 1, the refocused INEPT pulse sequence does not completely suppress unwanted 13CH3 signals. The presence of weak 13CH3 peaks is usually not a serious problem for smaller proteins because there are relatively few methyl signals and they are sharp; however, signal overlap becomes more common as the size of the protein increases. We overcome this problem by preparing a protein using a 98% D2O cell culture medium containing 3-(13)C pyruvic acid, 50-60% deuterated at the 3-position, and 4-(13)C 2-ketobutyric acid, 98% and 62% deuterated at the 3- and 4-positions, respectively. This approach significantly reduces the population of the CH3 isotopomer while optimizing the production of 13CHD2, the isotopomer desired for 13C relaxation measurements. In larger proteins where the deuterium T2 may be too short to measure accurately, we also suggest the alternative measurement of the proton T2 of the 13CH2D methyl isotopomer, because these protons are well-isolated from other protons in these highly deuterated samples.
journal_name
J Biomol NMRjournal_title
Journal of biomolecular NMRauthors
Ishima R,Louis JM,Torchia DAdoi
10.1023/a:1012482426306subject
Has Abstractpub_date
2001-10-01 00:00:00pages
167-71issue
2eissn
0925-2738issn
1573-5001journal_volume
21pub_type
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