Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins |
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Authors: | Jacob Jaison Louis John M Nesheiwat Issa Torchia Dennis A |
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Institution: | (1) Molecular Structural Biology Unit, National Institute of Dental and Craniofacial Research, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, U.S.A;(2) Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, U.S.A |
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Abstract: | Analysis of 2D 13C,1H]-HSQC spectra of biosynthetic fractionally 13C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional 13C labeling yields aromatic rings in which some of the 13C-13C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the 13C constant-time period in 2D 13C,1H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic 13C CSA and 13C-1H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution 13C constant-time spectra with good sensitivity. |
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Keywords: | amino acid aromatic NMR non-random 13C labeling proteins |
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