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Selective Protonation of Methyl Groups in Highly Deuterated Proteins

Vitali Tugarinov
Department of Chemistry and Biochemistry
Center for Biomolecular Structure and Organization
University of Maryland, College Park, Maryland 20742

Lewis E. Kay
Departments of Molecular Genetics, Biochemistry and Chemistry
University of Toronto
Toronto, Ontario, Canada M5S 1A8,
and Hospital for the Sick Children
Program in Molecular Structure and Function
555 University Avenue, Toronto, Ontario, Canada M5G 1X8

 

In structural NMR studies of small proteins, a maximum number of proton chemical shifts are usually assigned and NOEs connecting large numbers of sites are subsequently quantified in terms of distance restraints that are then used to obtain an ensemble of structures.1 For proteins exceeding ~50 kDa in molecular weight, this approach becomes less successful because sensitivity limitations and overlap of resonances preclude detailed analysis of all (protonated) sites in a protein. Therefore, in NMR studies of large proteins key positions are targeted for selective isotope labeling and protonation, while the remaining portions of the molecule are highly deuterated.2,3 Commonly, backbone amides and/or methyl groups are chosen for selective protonation and NMR analysis.3 In favorable cases, this subset of protonated sites provides a sufficient number of restraints to define a backbone global fold4,5 or characterize the structural and motional properties of a protein,6-8 while deuteration of the rest of the molecule significantly improves relaxation properties of the remaining subset of protons detected in NMR experiments.4,9,10 Due to their frequent location in the hydrophobic cores of protein structures and at molecular interfaces as well as favorable relaxation properties, methyl groups are especially useful NMR probes of structure and dynamics of large proteins.4,9,10 In the past decade, the strategy of limited protonation in an otherwise highly deuterated background that is exploited using 15N-1H- and methyl-TROSY techniques11,12 which isolate and preserve the slowly decaying components of NMR signals, had a significant impact on NMR studies of the structure and dynamics of large protein assemblies in the 100-800 kDa molecular weight range.7,8,13,14

Whereas 15N-1H moieties can be introduced into otherwise deuterated proteins via the use of 15N-labeled nitrogen sources followed by back-exchange of amide sites in H2O after protein production in D2O-based minimal media, selective protonation of methyl sites requires the use of appropriately labeled biosynthetic precursors and/or amino-acids during protein preparation. Although for NMR studies of smaller proteins, methyl-13C-labeled15 or [U-13C]-labeled2 pyruvate can be used as primary carbon sources for production of proteins protonated at Alaβ, Ileγ2, Valγ and Leuδ methyl sites in a highly deuterated background, this approach leads to extensive generation of methyl isotopomers of other than the 13CH3 variety (13CHD2, 13CH2D and 13CD3). Generation of methyl isotopomers degrades resolution and sensitivity of NMR spectra, and is very detrimental to the quality of NMR data obtained on larger systems. This is why the use of biosynthetic precursors/amino-acids with desired labeling patterns and with incorporation of labels of close to 100% is the strategy of choice for solution NMR studies of large proteins and macromolecular assemblies.

The development of strategies for selective methyl protonation of highly deuterated large proteins has a long history.9,10,16 Most important, the realization that α-ketoacids can serve as biosynthetic precursors has significantly impacted the development of isotope labeling strategies. Initial development of highly efficient methyl labeling techniques involved protonation of Ileδ1 positions through the addition of α-keto-butyrate (obtained by an enzymatic reaction from threonine) to D2O growth media.17 Later, it was shown that the addition of α-keto-isovalerate leads to efficient production of methyl-protonated Val and Leu residues.18 Despite the fact that the number of methyl-containing sites is limited to Ileδ1, Valγ, and Leuδ (ILV), these three residues comprise over 20% of the amino acid content of a ‘typical’ protein,19 and it has been shown that a large number of probes of structure and dynamics are available via this route. Building upon the efficiency and robustness of selective methyl protonation through the use of α-ketoacids, the Abbott group developed synthetic methods for production of α-keto-butyric and α-keto-isovaleric acids with 13C enrichment exclusively at methyl positions for high throughput screening of ligands to larger proteins.20 An alternative and more cost-effective synthetic strategy for production of these keto-acids was proposed later by Wagner and co-workers and used for studies of protein-ligand interactions.21 Subsequent developments included isopropyl 13CH3/12CD3 labeling (Val/Leu; one methyl group - 13CH3, the other - 12CD3), as described by Tugarinov and Kay22 and Konrat and co-workers.23 The latter group reported a versatile synthetic route for production of the [13CH3/12CD3]-labeled α-keto-isovalerate,23 although it is now available at reasonable prices commercially. It is important to note that this labeling strategy maximizes deuteration levels and optimizes the methyl-TROSY effect in Leu and Val side chains.22,24 Experimental details about ILV labeling protocols can be found in Tugarinov et al.25

The primary advantage of using α-keto-butyrate and α-keto-isovalerate precursors is that it is possible to incorporate many different labeling patterns into the side-chains of ILV residues. For example, using appropriate precursors the side-chains of ILV residues may be uniformly 13C-labeled or, alternatively, the 13C labels can be restricted to methyl groups. Likewise, methyl groups of the 13CH3, 13CHD2, or 13CH2D variety can be incorporated into ILV positions. Such labeling possibilities have greatly facilitated methyl-based NMR studies of large protein molecules. Tugarinov and Kay showed that using methyl-detected ‘out-and-back’ NMR experiments, assignments could be obtained for Leu and Val groups of monomeric proteins of up to ~100 kDa using ‘linearization’ of the corresponding spin-systems via α-keto-isovalerate labeled with 13C in only one of its two methyl positions.26 The utility of 13CHD2 and 13CH2D methyl isotopomers incorporated into the ILV side-chains via the use of [13CHD2 or 13CH2D]-labeled ketoacids has been established for 2H and 13C NMR relaxation studies of fast (pico- to nanosecond) side-chain motions.7,27-29 Comparisons of different methyl labeling strategies can be found in Ollerenshaw et al.30 and Religa and Kay.31 13CHD2 methyl isotopomers have recently found applications in relaxation dispersion studies of slow (milli- to micro-second) protein dynamics in very large protein assemblies.32

The ‘basic’ ILV labeling scheme described above has been augmented recently in a number of ways. One obvious shortcoming of Leu, Val labeling using [13CH3/12CD3]-α-keto-isovalerate is that the labels are incorporated into LV methyl groups non-stereospecifically, i.e. either pro-R or pro-S position is labeled in any single L/V side-chain. This leads to reduction of isotope incorporation (and sensitivity in 1H-13C methyl correlation maps) by a factor of 2 compared to the case when all the labels are concentrated at pro-R or pro-S sites. Stereospecific (pro-S) methyl labeling of LV side chains via the use of appropriately methyl-labeled aceto-lactate as a biosynthetic precursor has been demonstrated by Boisbouvier and co-workers;33 precursors for either pro-S or pro-R labeling are now available commercially. Although the use of aceto-lactate requires production of two separate protein samples (one labeled at pro-R and the other at pro-S methyl positions) and the precursors are considerably more expensive, this labeling approach leads to an approximate 2-fold increase in the sensitivity of methyl signals in 1H-13C methyl correlation maps, improved resolution and an approximate 4-fold sensitivity enhancement of contacts between Leu and Val methyls in NOE spectra of each protein sample. Further, it has been recently been shown by the Kay group that Ileγ2 methyls can be labeled using α-aceto-α-hydroxy-butyrate as a biosynthetic precursor (stable precursor is available commercially in ethyl-ester form).34 An efficient synthetic route has also been reported to produce α-aceto-α-hydroxy-butyrate for specific labeling of Ileγ2 positions.35 Both Ileγ2 labeling and stereospecific [13CH3/12CD3] labeling of Leu, Val methyls in a deuterated background are important extensions of ILV labeling methodology and will undoubtedly find multiple applications in NMR studies of supramolecular protein systems.  

Alaβ methyl groups are especially attractive probes of structure and dynamics in large proteins due to their proximity to the backbone and the resulting high degree of order. Alaβ methyl probes thus report on the properties of the protein backbone and hence provide important complementary information. A number of recent publications outline approaches for production of highly deuterated, {Alaβ-[13CH3]}-labeled proteins.36,37 Labeling of Ala methyls is challenging since this residue is produced directly as a result of transamination of pyruvate, which is also a precursor in the production of branched-chain amino acids. Transamination is reversible, i.e. if free methyl-labeled Ala is supplied to the medium, scrambling will occur, with the labels incorporated at a variety of potentially undesired locations. Boisbouvier and co-workers have developed a procedure to generate Ala methyl labeling with minimal (< 1%) scrambling.37 This was achieved by adding {2-[2H]; 3-[13C]}-Ala} (800 mg/L) together with deuterated precursors to ‘short-circuit’ the biosynthetic pathways that could lead to labeling at undesired positions.37 Several applications involving Ala methyl probes can be envisaged, including the measurement of backbone dynamics through relaxation studies, probing structure via residual dipolar couplings, methyl-methyl NOEs and studies of molecular interactions. Recently, Tugarinov and co-workers have demonstrated the utility and importance of Alaβ probes for NMR studies of large proteins from the perspective of both structure and dynamics using {Alaβ-[13CH3]}-, {Alaβ-[13CH3] + ILV-[13CH3/12CD3]}-, and {Alaβ-[13CHD2]}-labeled protein samples.38
   
Finally, due to the end position of methionine in the amino-acid biosynthetic pathways Met methyls can be incorporated into proteins by direct addition of [ε-13CH3]-labeled Met39 to bacterial cultures. Met labeling has been used in a number of NMR applications involving very large proteins and protein complexes.8,40

 

Materials List

     

References

  1. Wüthrich, K. NMR of Proteins and Nucleic Acids.; John Wiley and Sons: New York, 1986.
  2. Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-36.
  3. Tugarinov, V.; Hwang, P. M.; Kay, L. E. Annu. Rev. Biochem. 2004, 73, 107-46.
  4. Gardner, K. H.; Rosen, M. K.; Kay, L. E. Biochemistry 1997, 36, 1389-401.
  5. Tugarinov, V.; Choy, W. Y.; Orekhov, V. Y.; Kay, L. E. Proc. Natl. Acad. Sci. USA 2005, 102, 622-7.
  6. Sprangers, R.; Gribun, A.; Hwang, P. M.; Houry, W. A.; Kay, L. E. Proc. Natl. Acad. Sci. USA 2005, 102, 16678-83.
  7. Sprangers, R.; Kay, L. E. Nature 2007, 445, 618-22.
  8. Religa, T. L.; Sprangers, R.; Kay, L. E. Science 2010, 328, 98-102.
  9. Tugarinov, V.; Kay, L. E. Chembiochem 2005, 6, 1567-77.
  10. Ruschak, A. M.; Kay, L. E. J. Biomol. NMR 2010, 46, 75-87.
  11. Pervushin, K.; Riek, R.; Wider, G.; Wüthrich, K. Proc. Natl. Acad. Sci. USA 1997, 94, 12366-71.
  12. Tugarinov, V.; Hwang, P. M.; Ollerenshaw, J. E.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 10420-8.
  13. Fiaux, J.; Bertelsen, E. B.; Horwich, A. L.; Wüthrich, K. Nature 2002, 418, 207-21.
  14. Velyvis, A.; Yang, Y. R.; Schachman, H. K.; Kay, L. E. Proc. Natl. Acad. Sci. USA 2007, 104, 8815-20.
  15. Lee, A. L.; Urbauer, J. L.; Wand, A. J. J. Biomol. NMR 1997, 9, 437-40.
  16. Gardner, K. H.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406.
  17. Gardner, K. H.; Kay, L. E. J. Am. Chem. Soc. 1997, 119, 7599-600.
  18. Goto, N. K.; Gardner, K. H.; Mueller, G. A.; Willis, R. C.; Kay, L. E. J. Biomol. NMR 1999, 13, 369-74.
  19. Gerstein, M. J. Mol. Biol. 1997, 274, 562-76.
  20. Hajduk, P. J.; Augeri, D. J.; Mack, J.; Mendoza, R.; Yang, J.; Betz , S. F.; Fesik, S. W. J. Am. Chem. Soc. 2000, 122, 7898–904.
  21. Gross, J. D.; Gelev, V. M.; Wagner, G. J. Biomol. NMR 2003, 235-42.
  22. Tugarinov, V.; Kay, L. E. J. Biomol. NMR 2004, 28, 165-72.
  23. Lichtenecker, R.; Ludwiczek, M. L.; Schmid, W.; Konrat, R. J. Am. Chem. Soc. 2004, 126, 5348-9.
  24. Sprangers, R.; Velyvis, A.; Kay, L. E. Nat. Methods 2007, 4, 697-703.
  25. Tugarinov, V.; Kanelis, V.; Kay, L. E. Nat. Protocols 2006, 1, 749-54.
  26. Tugarinov, V.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 13868-78.
  27. Tugarinov, V.; Ollerenshaw, J. E.; Kay, L. E. J. Am. Chem. Soc. 2005, 127, 8214-25.
  28. Tugarinov, V.; Kay, L. E. Biochemistry 2005, 44, 15970-7.
  29. Sheppard, D.; Sprangers, R.; Tugarinov, V. Prog. Nucl. Magn. Reson. Spectrosc. 2010, 56, 1-45.
  30. Ollerenshaw, J. E.; Tugarinov, V.; Skrynnikov, N. R.; Kay, L. E. J. Biomol. NMR 2005, 33, 25-41.
  31. Religa, T. L.; Kay, L. E. J. Biomol. NMR 2010, 47, 163-9.
  32. Baldwin, A. J.; Religa, T. L.; Hansen, D. F.; Bouvignies, G.; Kay, L. E. J. Am. Chem. Soc. 2010, 132, 10992-5.
  33. Gans, P.; Hamelin, O.; Sounier, R.; Ayala, I.; Durá, M. A.; Amero, C. D.; Noirclerc-Savoye, M.; Franzetti, B.; Plevin, M. J.; Boisbouvier, J. Angew. Chem. Int. Ed. Engl. 2010, 49, 1958-62.
  34. Ruschak, A. M.; Velyvis, A.; Kay, L. E. J. Biomol. NMR 2010, 48, 129-35.
  35. Ayala, I.; Hamelin, O.; Amero, C.; Pessey, O.; Plevin, M. J.; Gans, P.; Boisbouvier, J. Chem. Commun. (Camb) 2011, DOI: 10.1039/C1CC12932E.
  36. Isaacson, R. L.; Simpson, P. J.; Liu, M.; Cota, E.; Zhang, X.; Freemont, P.; Matthews, S. J. Am. Chem. Soc. 2007, 129, 15428-9.
  37. Ayala, I.; Sounier, R.; Usé, N.; Gans, P.; Boisbouvier, J. J. Biomol. NMR 2009, 43., 111-9.
  38. Godoy-Ruiz, R.; Guo, C.; Tugarinov, V. J. Am. Chem. Soc. 2010, 132, 18340-50.
  39. Fischer, M.; Kloiber, K.; Häusler, J.; Ledolter, K.; Konrat, R.; Schmid, W. Chembiochem 2007, 8,  610-2.
  40. Gelis, I.; Bonvin, A. M.; Keramisanou, D.; Koukaki, M.; Gouridis, G.; Karamanou, S.; Economou, A.; Kalodimos, C. G. Cell 2007, 131, 756-69.

 

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