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Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl–methyl nuclear overhauser enhancement spectroscopy

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Abstract

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl–methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

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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 

  • Bernini A, Venditti V, Spiga O, Niccolai N (2009) Probing protein surface accessibility with solvent and paramagnetic molecules. Prog Nucl Magn Reson Spectrosc 54:278–289

    Article  Google Scholar 

  • Clore GM (2011) Exploring sparsely populated states of macromolecules by diamagnetic and paramagnetic NMR relaxation. Protein Sci 20:229–246

    Article  Google Scholar 

  • Clore GM, Iwahara J (2009) Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem Rev 109:4108–4139

    Article  Google Scholar 

  • Clore GM, Kuszewski J (2002) χ1 rotamer populations and angles of mobile surface side chains are accurately predicted by a torsion angle database potential of mean force. J Am Chem Soc 124:2866–2867

    Article  Google Scholar 

  • Fawzi NL, Fleissner MR, Anthis NJ, Kálai T, Hideg K, Hubbell WL, Clore GM (2011) A rigid, small, cysteine-reactive nitroxide spin label simplifies the quantitative analysis of PRE data. J Biomol NMR. doi:10:1007/s10858-011-9545-x

  • Fleissner MR, Brustad EM, Kalai T, Altenbach C, Cascio D, Peters FB, Hideg K, Peuker S, Schultz PG, Hubbell WL (2009) Site-directed spin labeling of a genetically encoded unnatural amino acid. Proc Natl Acad Sci USA 106:21637–21642

    Article  ADS  Google Scholar 

  • Gardner KH, Konrat R, Rosen MK, Kay LE (1996) An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated (15)N, (13)C-labeled proteins. J Biomol NMR 8:351–356

    Article  Google Scholar 

  • Garrett DS, Seok YJ, Liao DI, Peterkofsky A, Gronenborn AM, Clore GM (1997) Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system by multidimensional NMR. Biochemistry 36:2517–2530

    Article  Google Scholar 

  • Garrett DS, Seok YJ, Peterkofsky A, Gronenborn AM, Clore GM (1999) Solution structure of the 40,000 Mr phosphoryl transfer complex between the N-terminal domain of enzyme I and HPr. Nat Struct Biol 6:166–173

    Article  Google Scholar 

  • Gelis I, Bonvin AM, Keramisanou D, Koukaki M, Gouridis G, Karamanou S, Economou A, Kalodimos CG (2007) Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131:756–769

    Article  Google Scholar 

  • Godoy-Ruiz R, Guo C, Tugarinov V (2010) Alanine methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins. J Am Chem Soc 132:18340–18350

    Article  Google Scholar 

  • Gross JD, Gelev VM, Wagner G (2003) A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes. J Biomol NMR 25:235–242

    Article  Google Scholar 

  • Hajduk P, Augeri D, Mack J, Mendoza R, Yang J, Betz S, Fesik S (2000) NMR-based screening of proteins containing 13C-labeled methyl groups. J Am Chem Soc 122:7898–7904

    Article  Google Scholar 

  • Iwahara J, Schwieters CD, Clore GM (2004) Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. J Am Chem Soc 126:5879–5896

    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 129:13749–13757

    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 

  • Metropolis N, Rosenbluth AW, Rosenbluth MN, Teller AH, Teller E (1953) Equations of state calculations by fast computing machines. J Chem Phys 21:1087–1092

    Article  ADS  Google Scholar 

  • Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371

    Article  ADS  Google Scholar 

  • Pintacuda G, Keniry MA, Huber T, Park AY, Dixon NE, Otting G (2004) Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins. J Am Chem Soc 126:2963–2970

    Article  Google Scholar 

  • Religa TL, Sprangers R, Kay LE (2010) Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR. Science 328:98–102

    Article  ADS  Google Scholar 

  • Schwieters CD, Clore GM (2002) Reweighted atomic densities to represent ensembles of NMR structures. J Biomol NMR 23:221–225

    Article  Google Scholar 

  • Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160:65–73

    Article  ADS  Google Scholar 

  • Schwieters CD, Suh JY, Grishaev A, Ghirlando R, Takayama Y, Clore GM (2010) Solution structure of the 128 kDa enzyme I dimer from Escherichia coli and its 146 kDa complex with HPr using residual dipolar couplings and small- and wide-angle X-ray scattering. J Am Chem Soc 132:13026–13045

    Article  Google Scholar 

  • Solomon I, Bloembergen N (1956) Nuclear magnetic interactions in the HF molecule. J Chem Phys 25:261–266

    Article  ADS  Google Scholar 

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

    Article  Google Scholar 

  • Tang C, Iwahara J, Clore GM (2006) Visualization of transient encounter complexes in protein–protein association. Nature 444:383–386

    Article  ADS  Google Scholar 

  • Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114

    Article  ADS  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, Hwang PM, Ollerenshaw JE, Kay LE (2003) Cross-correlated relaxation enhanced 1H-13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J Am Chem Soc 125:10420–10428

    Article  Google Scholar 

  • Tugarinov V, Choy WY, Orekhov VY, Kay LE (2005) Solution NMR-derived global fold of a monomeric 82-kDa enzyme. Proc Natl Acad Sci USA 102:622–627

    Article  ADS  Google Scholar 

  • Tugarinov V, Kanelis V, Kay LE (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc 1:749–754

    Article  Google Scholar 

  • Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J, Livny M, Mading S, Maziuk D, Miller Z, Nakatani E, Schulte CF, Tolmie DE, Kent Wenger R, Yao H, Markley JL (2008) BioMagResBank. Nucleic Acids Res 36:D402–D408

    Article  Google Scholar 

  • Velyvis A, Yang YR, Schachman HK, Kay LE (2007) A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase. Proc Natl Acad Sci USA 104:8815–8820

    Article  ADS  Google Scholar 

  • Vuister GM, Clore GM, Gronenborn AM, Powers R, Garrett DS, Tschudin R, Bax A (1993) Increased resolution and improved spectral quality in four-dimentional 13C/13C separated HMQC-NOESY-HMQC spectra using pulsed field gradients. J Magn Reson Ser B 101:210–213

    Google Scholar 

  • Xu Y, Liu M, Simpson PJ, Isaacson R, Cota E, Marchant J, Yang D, Zhang X, Freemont P, Matthews S (2009) Automated assignment in selectively methyl-labeled proteins. J Am Chem Soc 131:9480–9481

    Article  Google Scholar 

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Acknowledgments

We thank Drs. Mark Fleissner, Kálmán Hideg, Tamás Kálai and Wayne Hubbell for generously providing the reagent for generating the R1p paramagnetic side chain. This work was supported by funds from the Intramural Program of the NIH, NIDDK, and the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the NIH (to G.M.C.).

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Authors and Affiliations

  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA

    Vincenzo Venditti, Nicolas L. Fawzi & G. Marius Clore

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  1. Vincenzo Venditti
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  2. Nicolas L. Fawzi
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  3. G. Marius Clore
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Correspondence to G. Marius Clore.

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Venditti, V., Fawzi, N.L. & Clore, G.M. 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 (2011). https://doi.org/10.1007/s10858-011-9559-4

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