Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins.
Selective incorporation of 13C into the methyl groups of protein side chains is described as a means for simplifying the measurement and interpretation of 13C relaxation parameters. High incorporation (> 90%) is accomplished by using pyruvate (3-13C, 99%) as the sole carbon source in the growth media for protein overexpression in E. coli. This improved labeling scheme increases the sensitivity of the relaxation experiments by approximately fivefold when compared to randomly fractionally 13C-labeled protein, allowing high-quality measurements on relatively dilute (< 1 mM) protein samples at a relatively low cost.
Lee
A L
AL
Department of Biological Sciences, State University of New York at Buffalo 14260-3000, USA.
Urbauer
J L
JL
Wand
A J
AJ
D001426:Bacterial Proteins / Q000096:biosynthesis; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008745:Methylation; D019289:Pyruvic Acid / Q000378:metabolism*; D014452:Ubiquitins / Q000096:biosynthesis* / Q000737:chemistry
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D014452:Ubiquitins; D019289:Pyruvic Acid