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. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of (1)H-(15)N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D (1)H-(15)N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected 'out-and-back' HMCM(CG)CBCA experiment, and (3) strong sequential HN-HN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578-195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.

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. Using methyl-based NMR spectroscopy, we provide evidence for symmetry in the absence of ligands in several homodimeric proteins that are either asymmetric functionally and/or adopt different conformations of the two subunits in available X-ray structures.