An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated (15)N, (13)C-labeled proteins.
A biosynthetic strategy has recently been developed for the production of (15)N, (13)C, (2)H-labeled proteins using (1)H(3)C-pyruvate as the sole carbon source and D(2)O as the solvent. The methyl groups of Ala, Val, Leu and Ile (gamma2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a (15)N, (13)C, (2)H, (1)H(3)C-labeled SH2 domain at 3 degrees C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30-40 kDa molecular weight range.
Gardner
K H
KH
The Protein Engineering Centers of Excellence, University of Toronto, M5S 1A8, Toronto, ON, Canada.
Konrat
R
R
Rosen
M K
MK
Kay
L E
LE