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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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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-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 1H–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 1H–15N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D 1H–15N 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.

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References

  • Boyd J, Soffe N (1989) Selective excitation by pulse shaping combined with phase modulation. J Magn Reson 85:406–413

    Google Scholar 

  • Brick P, Blow DM (1987) Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine. J Mol Biol 194:287–297

    Article  Google Scholar 

  • Brick P, Bhat TN, Blow DM (1989) Structure of tyrosyl-tRNA synthetase refined at 2.3A resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate. J Mol Biol 208:83–98

    Article  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  Google Scholar 

  • Fersht AR (1975) Demonstration of two active sites on a monomeric aminoacyl-tRNA synthetase. Possible roles of negative cooperativity and half-of-the-sites reactivity in oligomeric enzymes. Biochemistry 14:5–12

    Article  Google Scholar 

  • Fersht AR (1987) Dissection of the structure and activity of the tyrosyl-tRNA synthetase by site-directed mutagenesis. Biochemistry 26:8031–8037

    Article  Google Scholar 

  • Fersht A (2002) Structure and mechanism in protein science. Freeman & Co., New York

    Google Scholar 

  • Gardner KH, Rosen MK, Kay LE (1997) Global folds of highly deuterated, methyl protonated proteins by multidimensional NMR. Biochemistry 36:1389–1401

    Article  Google Scholar 

  • Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141

    Google Scholar 

  • Godoy-Ruiz R, Krejcirikova A, Gallagher DT, Tugarinov V (2011) Solution NMR evidence for symmetry in functionally or crystallographically asymmetric homodimers. J Am Chem Soc 133:19578–19581

    Article  Google Scholar 

  • Guijarro JI, Pintar A, Prochnicka-Chalufour A, Guez V, Gilquin B, Bedouelle H, Delepierre M (2002) Structure and dynamics of the anticodon arm binding domain of Bacillus stearothermophilus Tyrosyl-tRNA synthetase. Structure 10:311–317

    Article  Google Scholar 

  • Johnson BA, Blevins RA (1994) NMRView: a computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614

    Article  Google Scholar 

  • Kay LE, Ikura M, Tschudin R, Bax A (1990) Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514

    Google Scholar 

  • Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665

    Article  Google Scholar 

  • Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J Magn Reson 85:393–399

    Google Scholar 

  • Park YC, Bedouelle H (1998) Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions. J Biol Chem 273:18052–18059

    Article  Google Scholar 

  • Patt SL (1992) Single- and multiple-frequency-shifted laminar pulses. J Magn Reson 96:94–102

    Google Scholar 

  • Salzmann M, Wider G, Pervushin K, Senn H, Wüthrich K (1999) TROSY-type triple resonance experiments for sequential NMR assignment of large proteins. J Am Chem Soc 121:844–848

    Article  Google Scholar 

  • Schleucher J, Sattler M, Griesinger C (1993) Coherence selection by gradients without signal attenuation: application to the three-dimensional HNCO experiment. Angew Chem Int Ed Engl 32:1489–1491

    Article  Google Scholar 

  • Spera S, Bax A (1991) Empirical correlation between protein backbone conformation and Cα and Cβ 13C nuclear magnetic resonance chemical shifts. J Am Chem Soc 113:5490–5492

    Article  Google Scholar 

  • Tugarinov V, Kay LE (2003) Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J Am Chem Soc 125:13868–13878

    Article  Google Scholar 

  • Tugarinov V, Muhandiram R, Ayed A, Kay LE (2002) Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase G. J Am Chem Soc 124:10025–10035

    Article  Google Scholar 

  • Venters RA, Farmer BT, Fierke CA, Spicer LD (1996) Characterizing the use of perdeuteration in NMR Studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II. J Mol Biol 264:1101–1116

    Article  Google Scholar 

  • Wagner G, Pardi A, Wüthrich K (1983) Hydrogen bond length and proton NMR chemical shifts in proteins. J Am Chem Soc 105:5948–5957

    Article  Google Scholar 

  • Ward WH, Fersht AR (1988a) Asymmetry of tyrosyl-tRNA synthetase in solution. Biochemistry 27:1041–1049

    Article  Google Scholar 

  • Ward WH, Fersht AR (1988b) Tyrosyl-tRNA synthetase acts as an asymmetric dimer in charging tRNA. A rationale for half-of-the-sites activity. Biochemistry 27:5525–5530

    Article  Google Scholar 

  • Waye MM, Winter G, Wilkinson AJ, Fersht AR (1983) Deletion mutagenesis using an ‘M13 splint’: the N-terminal structural domain of tyrosyl-tRNA synthetase (B. stearothermophilus) catalyses the formation of tyrosyl adenylate. EMBO J 2:1827–1829

    Google Scholar 

  • Williamson MP (1990) Secondary-structure dependent chemical shifts in proteins. Biopolymers 29:1423–1431

    Article  Google Scholar 

  • Wishart DS, Case DA (2002) Use of chemical shifts in macromolecular structure determination. Methods Enzymol 338:3–34

    Article  Google Scholar 

  • Wishart DS, Sykes BD (1994a) The 13C chemical-shift index: a simple method for identification of protein secondary structure using 13C chemical shift data. J Biomol NMR 4:171–180

    Article  Google Scholar 

  • Wishart DS, Sykes BD (1994b) Chemical shifts as a tool for structure determination. Methods Enzymol 239:363

    Article  Google Scholar 

  • Wishart DS, Sykes BD, Richards FM (1992) The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31:1647–1651

    Article  Google Scholar 

  • Yang D, Kay LE (1999) TROSY triple resonance four-dimensional NMR spectroscopy of a 46 ns tumbling protein. J Am Chem Soc 121:2571–2575

    Article  Google Scholar 

Download references

Acknowledgments

The authors thank Prof. Paul Paukstelis (University of Maryland) for the gift of the plasmid encoding full-length Bst-YRS from which the plasmid for ΔYRS expression was derived, and Prof. Eric First (Louisiana State University Health Center) for stimulating discussions. Backbone and ILV methyl assignments of Bst-ΔYRS have been deposited to BMRB data bank, http://www.bmrb.wisc.edu, entry #18588.

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  1. Department of Chemistry and Biochemistry, University of Maryland, Biomolecular Sci. Bldg./CBSO, College Park, MD, 20742, USA

    Anna Krejcirikova & Vitali Tugarinov

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  1. Anna Krejcirikova
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  2. Vitali Tugarinov
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Correspondence to Vitali Tugarinov.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10858_2012_9667_MOESM1_ESM.pdf

A table containing 1HN, 15N, 13Cα, 13Cβ, 13CO and 1H, 13C chemical shifts of Ileδ1, Leuδ and Valγ methyls of Bst-ΔYRS (pH = 6.8, 40 °C). A listing of the Bruker code used for the Ala- and Thr/Ser – TROSY-HN(CACB) experiment. (PDF 38 kb)

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Krejcirikova, A., Tugarinov, V. 3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample. J Biomol NMR 54, 135–143 (2012). https://doi.org/10.1007/s10858-012-9667-9

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