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Divide and conquer is always best: sensitivity of methyl correlation experiments

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Abstract

The HMCM [CG]CBCA experiment (Tugarinov and Kay in J Am Chem Soc 125:13868–13878, 2003) correlates methyl carbon and proton shifts to Cγ, Cβ, and Cα resonances for the purpose of resonance assignments. The relative sensitivity of the HMCM[CG]CBCA sequence experiment is compared to a divide-and-conquer approach to assess whether it is best to collect all of the methyl correlations at once, or to perform separate experiments for each correlation. A straightforward analysis shows that the divide-and-conquer approach is intrinsically more sensitive, and should always be used to obtain methyl-Cγ, Cβ, and Cα correlations. The improvement in signal-to-noise associated with separate experiments is illustrated by the detection of methyl-aliphatic correlations in a 65 kDa protein-DNA complex.

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References

  • Ayala I, Sounier R, Use N, Gans P, Boisbouvier J (2009) An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR 43:111–119

    Article  Google Scholar 

  • Baldwin AJ, Religa TL, Hansen DF, Bouvignies G, Kay LE (2010) 13CHD2 methyl group probes of millisecond time scale exchange in proteins by 1H relaxation dispersion: an application to proteasome gating residue dynamics. J Am Chem Soc 132(32):10992–10995. doi:10.1021/ja104578n

    Article  Google Scholar 

  • Biswas S, Guharoy M, Chakrabarti P (2009) Dissection, residue conservation, and structural classification of protein-DNA interfaces. Proteins 74:643–654

    Article  Google Scholar 

  • Briercheck DM, Wood TC, Allison TJ, Richardson JP, Rule GS (1998) The NMR structure of the RNA binding domain of E. coli rho factor suggests possible RNA-protein interactions. Nat Struct Biol 5:393–399

    Article  Google Scholar 

  • Chan PH, Weissbach S, Okon M, Withers SG, McIntosh LP (2012) Nuclear magnetic resonance spectral assignments of α-1,4-galactosyltransferase LgtC from neisseria meningitidis: substrate binding and multiple conformational states. Biochemistry 51:8278–8292

    Article  Google Scholar 

  • Fernandez C, Wider G (2003) TROSY in NMR studies of the structure and function of large biological macromolecules. Curr Opin Struct Biol 13:570–580

    Article  Google Scholar 

  • Gardner KH, Zhang X, Gehring K, Kay LE (1998) Solution NMR Studies of a 42 KDa Escherichia Coli Maltose Binding Protein/β-Cyclodextrin Complex: chemical Shift Assignments and Analysis. J Am Chem Soc 120(45):11738–11748. doi:10.1021/ja982019w

    Article  Google Scholar 

  • Göbl C, Tjandra N (2012) Application of solution NMR spectroscopy to study protein dynamics. Entropy 14(3):581–598. doi:10.3390/e14030581

    Article  Google Scholar 

  • Goto NK, Kay LE (2000) New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr Opin Struct Biol 10:585–592

    Article  Google Scholar 

  • Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J Biomol NMR 13:369–374

    Article  Google Scholar 

  • John M, Schmitz C, Park AY, Dixon NE, Huber T, Otting G (2007) Sequence-specific and stereospecific assignment of methyl groups using paramagnetic lanthanides. J Am Chem Soc 7:13749–13757

    Article  Google Scholar 

  • Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta. doi:10.1016/j.bbapap.2010.10.012

    Google Scholar 

  • Krejcirikova A, Tugarinov V (2012) 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. doi:10.1007/s10858-012-9667-9

    Article  Google Scholar 

  • LeMaster DM, Richards FM (1988) NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. Biochemistry 27:142–150

    Article  Google Scholar 

  • Markwick PR, Malliavin T, Nilges M (2008) Structural biology by NMR: structure, dynamics, and interactions. PLoS Comput Biol 4(9):e1000168. doi:10.1371/journal.pcbi.1000168

    Article  Google Scholar 

  • McCallum SA, Hitchens TK, Rule GS (1999) Solution structure of the carboxyl terminus of a human class mu glutathione s-transferase: NMR assignment strategies in large proteins. J Mol Biol 285:2119–2132

    Article  Google Scholar 

  • Mittermaier A, Kay LE (2006) New tools provide new insights in NMR studies of protein dynamics. Science 312:224–228

    Article  ADS  Google Scholar 

  • Mueller GA, Choy WY, Yang D, Forman-Kay JD, Venters RA, Kay LE (2000) Global folds of proteins with low densities of noes using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein. J Mol Biol 300:197–212

    Article  Google Scholar 

  • Ruschak AM, Kay LE (2010) Methyl groups as probes of supra-molecular structure, dynamics and function. J Biomol NMR 46:75–87. doi:10.1007/s10858-009-9376-1

    Article  Google Scholar 

  • Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci U S A. 95(23):13585–13590

    Article  ADS  Google Scholar 

  • Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (2000) NMR Assignment and Secondary Structure Determination of an Octameric 110 kDa Protein Using TROSY in Triple Resonance Experiments. J Am Chem Soc 122:7543–7548. doi:10.1021/ja0003268

    Article  Google Scholar 

  • Sattler M, Fesik SW (1996) Use of deuterium labeling in NMR: overcoming a sizeable problem. Structure 4:1245–1249

    Article  Google Scholar 

  • Shajani Z, Varani G (2007) NMR studies of dynamics in RNA and DNA by 13C relaxation. Biopolymers 86:348–359. doi:10.1002/bip.20650

    Article  Google Scholar 

  • Sinha K, Jen-Jacobson L, Rule GS (2011) Specific labeling of threonine methyl groups for NMR studies of protein-nucleic acid complexes. Biochemistry 50:10189–10191. doi:10.1021/bi201496d

    Article  Google Scholar 

  • Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–622. doi:10.1038/nature05512

    Article  Google Scholar 

  • Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234

    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. doi:10.1021/ja030345s

    Article  Google Scholar 

  • Tugarinov V, Kay LE (2005) Quantitative 13C and 2H NMR relaxation studies of the 723-residue enzyme malate synthase G reveal a dynamic binding interface. Biochemistry 44:15970–15977. doi:10.1021/bi0519809

    Article  Google Scholar 

  • Tzakos AG, Grace CRR, Lukavsky PJ, Riek R (2006) NMR Techniques for Very Large Proteins and RNAs in Solution. Annu Rev Biophys Biomol Struct 35:319–342

    Article  Google Scholar 

  • Velyvis A, Ruschak AM, Kay LE (2012) An economical method for production of (2)H, (13)CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome. PLoS ONE 7(9):e43725. doi:10.1371/journal.pone.0043725

    Article  ADS  Google Scholar 

  • Venditti V, Fawzi NL, Clore GM (2011) Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl–methyl nuclear Overhauser enhancement spectroscopy. J Biomol NMR 51:319–328

    Article  Google Scholar 

  • Wang X, Vu A, Lee K, Dahlquist FW (2012) CheA–Receptor Interaction Sites in Bacterial Chemotaxis. J Mol Biol 422:282–290. doi:10.1016/j.jmb.2012.05.023

    Article  Google Scholar 

  • Xu Y, Matthews S (2013) MAP-XSII: an improved program for the automatic assignment of methyl resonances in large proteins. J Biomol NMR 55:179–187. doi:10.1007/s10858-012-9700-z

    Article  Google Scholar 

  • Zhuravleva A, Clerico EM, Gierasch LM (2012) An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones. Cell 151:1296–1307

    Article  Google Scholar 

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Acknowledgments

We thank Virgil Simplaceanu for maintenance of the NMR spectrometers and Dr. C. Sanders (Vanderbilt University) for providing the Bruker code for the HMCM[CG]CBCA experiment. This work was supported by the Mellon College of Science at CMU and an NIH Merit grant 5R37-GM029207 to L.J–J.

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Authors and Affiliations

  1. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, 15213, USA

    Kaustubh Sinha & Gordon S. Rule

  2. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, 15213, USA

    Linda Jen-Jacobson

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  1. Kaustubh Sinha
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  2. Linda Jen-Jacobson
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Correspondence to Gordon S. Rule.

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Sinha, K., Jen-Jacobson, L. & Rule, G.S. Divide and conquer is always best: sensitivity of methyl correlation experiments. J Biomol NMR 56, 331–335 (2013). https://doi.org/10.1007/s10858-013-9751-9

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