Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins.
Analysis of 2D [(13)C,(1)H]-HSQC spectra of biosynthetic fractionally (13)C 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 (13)C labeling yields aromatic rings in which some of the (13)C-(13)C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the delta-, epsilon- and zeta-carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the (13)C constant-time period in 2D [(13)C,(1)H]-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 (13)C CSA and (13)C-(1)H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution (13)C constant-time spectra with good sensitivity.
Jacob
Jaison
J
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, USA.
Louis
John M
JM
Nesheiwat
Issa
I
Torchia
Dennis A
DA
D024322:Amino Acids, Aromatic / Q000737:chemistry*; D002247:Carbon Isotopes / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010449:Peptide Mapping; D010649:Phenylalanine / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods; D014443:Tyrosine / Q000737:chemistry
D024322:Amino Acids, Aromatic; D002247:Carbon Isotopes; D011506:Proteins; D014443:Tyrosine; D010649:Phenylalanine