Abstract The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-2H,15N,13C; Ileδ1-{13CH3}; Leu,Val-{13CH3/12CD3}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-Î?YRS), 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 1Hâ??15N 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 1Hâ??15N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D 1Hâ??15N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected â??out-and-backâ?? HMCM(CG)CBCA experiment, and (3) strong sequential HNâ??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 133:19578â??195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.
Content Type Journal Article
Category Communication
Pages 1-9
DOI 10.1007/s10858-012-9667-9
Authors
Anna Krejcirikova, Department of Chemistry and Biochemistry, University of Maryland, Biomolecular Sci. Bldg./CBSO, College Park, MD 20742, USA
Vitali Tugarinov, Department of Chemistry and Biochemistry, University of Maryland, Biomolecular Sci. Bldg./CBSO, College Park, MD 20742, USA
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3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample
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