Selective methyl group protonation of perdeuterated proteins.
Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy. One problem inherent in high-level deuterium incorporation is the loss of 1H-1H distance information obtainable from NOESY spectra of the labeled proteins. In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein. We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples. Overexpression of proteins in bacteria grown in 2H2O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C alpha and > 80% deuteration at C beta positions of nearly all amino acids. In contrast, the methyl groups of Ala, Val, Leu and Ile (gamma 2 only) remain highly protonated. This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis. As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR.
Rosen
M K
MK
Department of Medical Genetics, University of Toronto, Ontario, Canada.
Gardner
K H
KH
Willis
R C
RC
Parris
W E
WE
Pawson
T
T
Kay
L E
LE
D000596:Amino Acids / Q000096:biosynthesis; D001419:Bacteria / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D019289:Pyruvic Acid / Q000378:metabolism
D000596:Amino Acids; D011506:Proteins; D011522:Protons; D019289:Pyruvic Acid; D003903:Deuterium