NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation.
All non-proline residues except for the N-terminal dipeptide have been assigned in the 108-residue protein Escherichia coli thioredoxin. Central to these experiments has been the use of protein samples in which all carbon-bound hydrogen positions are substituted to 75% with deuterium by bacterial growth on partially deuteriated carbon sources and media. The dilution of the local proton density gives rise to narrower line widths with little loss in sensitivity. In addition, passive or secondary coupling to protons not directly involved in the coherence transfer process of correlation experiments is largely suppressed, thus significantly improving the resolution for side-chain couplings. Simultaneous multiresidue-type assignments have been obtained by incorporation of several amino acids with differing selective alpha- and/or beta-deuteriation into a fractionally deuteriated background. Combined with several single residue type labeling experiments, these selective labelings have yielded direct residue type assignments for two-thirds of the protein. In addition to improved resolution, the amide to carbon-bound proton NOESY spectra offered equivalent sensitivity while the amide to amide NOESY spectra offered superior sensitivity to that observed for natural abundance samples. The resultant sequential assignment has an average number of nearest-neighbor NOE connectivities of 2.35 out of the possible 3 alpha-amide, beta-amide, and amide-amide connectivities.
LeMaster
D M
DM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Richards
F M
FM
D001426:Bacterial Proteins / Q000235:genetics; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010957:Plasmids; D011487:Protein Conformation; D013604:T-Phages / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics
D001426:Bacterial Proteins; D013879:Thioredoxins; D003903:Deuterium