A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins.
A selective protonation strategy is described that uses [3-2H] 13C alpha-ketoisovalerate to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of approximately 100 mg/L alpha-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] alpha-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-delta1 methyl)-isoleucine (> 90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
Goto
N K
NK
Department of Biochemistry, University of Toronto, ON, Canada. gnat@pound.med.utoronto.ca
Gardner
K H
KH
Mueller
G A
GA
Willis
R C
RC
Kay
L E
LE
D002247:Carbon Isotopes; D003903:Deuterium; D007202:Indicators and Reagents; D007532:Isoleucine; D007553:Isotope Labeling / Q000379:methods*; D007930:Leucine; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry*; D014633:Valine
D002247:Carbon Isotopes; D007202:Indicators and Reagents; D009587:Nitrogen Isotopes; D011506:Proteins; D007532:Isoleucine; D003903:Deuterium; D007930:Leucine; D014633:Valine