3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample.

The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-(2)H,(15)N,(13)C; Ileδ1-{(13)CH(3)}; Leu,Val-{(13)CH(3)/(12)CD(3)}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-ΔYRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40 °C to improve the sensitivity of the measurements and alleviate the overlap of (1)H-(15)N correlations in the abundant α-helical segments of the protein.

Solution NMR evidence for symmetry in functionally or crystallographically asymmetric homodimers.

A recurrent theme of many structural studies of homo-oligomeric protein systems is concerned with verification that the conformation observed in a crystal represents the functionally relevant structure. An asymmetric conformation adopted by two chemically identical subunits in homo-oligomers can represent an intrinsic property of a protein or be an artifact induced by crystal packing forces. Solution NMR studies can distinguish between these two possibilities.