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
Loading

References

1.
Wüthrich K. 1986. NMR with Proteins and Nucleic Acids. Europhys. News. 17(1):11-13. http://dx.doi.org/10.1051/epn/19861701011
2.
Rosen MK, Gardner KH, Willis RC, Parris WE, Pawson T, Kay LE. 1996. Selective Methyl Group Protonation of Perdeuterated Proteins. Journal of Molecular Biology. 263(5):627-636. http://dx.doi.org/10.1006/jmbi.1996.0603
3.
Tugarinov V, Hwang PM, Kay LE. 2004. Nuclear Magnetic Resonance Spectroscopy of High-Molecular-Weight Proteins. Annu. Rev. Biochem.. 73(1):107-146. http://dx.doi.org/10.1146/annurev.biochem.73.011303.074004
4.
Gardner KH, Rosen MK, Kay LE. 1997. Global Folds of Highly Deuterated, Methyl-Protonated Proteins by Multidimensional NMR?. Biochemistry. 36(6):1389-1401. http://dx.doi.org/10.1021/bi9624806
5.
Tugarinov V, Choy W, Orekhov VY, Kay LE. 2005. Solution NMR-derived global fold of a monomeric 82-kDa enzyme. Proceedings of the National Academy of Sciences. 102(3):622-627. http://dx.doi.org/10.1073/pnas.0407792102
6.
Sprangers R, Gribun A, Hwang PM, Houry WA, Kay LE. 2005. Quantitative NMR spectroscopy of supramolecular complexes: Dynamic side pores in ClpP are important for product release. Proceedings of the National Academy of Sciences. 102(46):16678-16683. http://dx.doi.org/10.1073/pnas.0507370102
7.
Sprangers R, Kay LE. 2007. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature. 445(7128):618-622. http://dx.doi.org/10.1038/nature05512
8.
Religa TL, Sprangers R, Kay LE. 2010. Dynamic Regulation of Archaeal Proteasome Gate Opening As Studied by TROSY NMR. Science. 328(5974):98-102. http://dx.doi.org/10.1126/science.1184991
9.
Tugarinov V, Kay LE. 2005. Methyl Groups as Probes of Structure and Dynamics in NMR Studies of High-Molecular-Weight Proteins. ChemBioChem. 6(9):1567-1577. http://dx.doi.org/10.1002/cbic.200500110
10.
Ruschak AM, Kay LE. 2010. Methyl groups as probes of supra-molecular structure, dynamics and function. J Biomol NMR. 46(1):75-87. http://dx.doi.org/10.1007/s10858-009-9376-1
11.
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. Proceedings of the National Academy of Sciences. 94(23):12366-12371. http://dx.doi.org/10.1073/pnas.94.23.12366
12.
Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE. 2003. Cross-Correlated Relaxation Enhanced1H?13C NMR Spectroscopy of Methyl Groups in Very High Molecular Weight Proteins and Protein Complexes. J. Am. Chem. Soc.. 125(34):10420-10428. http://dx.doi.org/10.1021/ja030153x
13.
Fiaux J, Bertelsen EB, Horwich AL, Wüthrich K. 2002. NMR analysis of a 900K GroEL?GroES complex. Nature. 418(6894):207-211. http://dx.doi.org/10.1038/nature00860
14.
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. Proceedings of the National Academy of Sciences. 104(21):8815-8820. http://dx.doi.org/10.1073/pnas.0703347104
15.
Lee AL, Urbauer JL, Wand AJ. 1997. 9(4):437-440. http://dx.doi.org/10.1023/a:1018311013338
16.
Gardner KH, Kay LE. 1998. THE USE OF2H,13C,15N MULTIDIMENSIONAL NMR GTO STUDY THE STRUCTURE AND DYNAMICS OF PROTEINS. Annu. Rev. Biophys. Biomol. Struct.. 27(1):357-406. http://dx.doi.org/10.1146/annurev.biophys.27.1.357
17.
Gardner KH, Kay LE. 1997. Production and Incorporation of15N,13C,2H (1H-?1 Methyl) Isoleucine into Proteins for Multidimensional NMR Studies. J. Am. Chem. Soc.. 119(32):7599-7600. http://dx.doi.org/10.1021/ja9706514
18.
Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE. 1999. 13(4):369-374. http://dx.doi.org/10.1023/a:1008393201236
19.
Gerstein M. 1997. A structural census of genomes: comparing bacterial, eukaryotic, and archaeal genomes in terms of protein structure. Journal of Molecular Biology. 274(4):562-576. http://dx.doi.org/10.1006/jmbi.1997.1412
20.
Hajduk PJ, Augeri DJ, Mack J, Mendoza R, Yang J, Betz SF, Fesik SW. 2000. NMR-Based Screening of Proteins Containing13C-Labeled Methyl Groups. J. Am. Chem. Soc.. 122(33):7898-7904. http://dx.doi.org/10.1021/ja000350l
21.
Gross JD, Gelev VM, Wagner G. 2003. 25(3):235-242. http://dx.doi.org/10.1023/a:1022890112109
22.
Tugarinov V, Kay LE. 2004. An Isotope Labeling Strategy for Methyl TROSY Spectroscopy. J Biomol NMR. 28(2):165-172. http://dx.doi.org/10.1023/b:jnmr.0000013824.93994.1f
23.
Lichtenecker R, Ludwiczek ML, Schmid W, Konrat R. 2004. Simplification of Protein NOESY Spectra Using Bioorganic Precursor Synthesis and NMR Spectral Editing. J. Am. Chem. Soc.. 126(17):5348-5349. http://dx.doi.org/10.1021/ja049679n
24.
Sprangers R, Velyvis A, Kay LE. 2007. Solution NMR of supramolecular complexes: providing new insights into function. Nat Methods. 4(9):697-703. http://dx.doi.org/10.1038/nmeth1080
25.
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(2):749-754. http://dx.doi.org/10.1038/nprot.2006.101
26.
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(45):13868-13878. http://dx.doi.org/10.1021/ja030345s
27.
Tugarinov V, Ollerenshaw JE, Kay LE. 2005. Probing Side-Chain Dynamics in High Molecular Weight Proteins by Deuterium NMR Spin Relaxation:  An Application to an 82-kDa Enzyme. J. Am. Chem. Soc.. 127(22):8214-8225. http://dx.doi.org/10.1021/ja0508830
28.
Tugarinov V, Kay LE. 2005. Quantitative13C and2H NMR Relaxation Studies of the 723-Residue Enzyme Malate Synthase G Reveal a Dynamic Binding Interface?. Biochemistry. 44(49):15970-15977. http://dx.doi.org/10.1021/bi0519809
29.
Sheppard D, Sprangers R, Tugarinov V. 2010. Experimental approaches for NMR studies of side-chain dynamics in high-molecular-weight proteins. Progress in Nuclear Magnetic Resonance Spectroscopy. 56(1):1-45. http://dx.doi.org/10.1016/j.pnmrs.2009.07.004
30.
Ollerenshaw JE, Tugarinov V, Skrynnikov NR, Kay LE. 2005. Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins. J Biomol NMR. 33(1):25-41. http://dx.doi.org/10.1007/s10858-005-2614-2
31.
Religa TL, Kay LE. 2010. Optimal methyl labeling for studies of supra-molecular systems. J Biomol NMR. 47(3):163-169. http://dx.doi.org/10.1007/s10858-010-9419-7
32.
Baldwin AJ, Religa TL, Hansen DF, Bouvignies G, Kay LE. 2010. 13CHD2Methyl Group Probes of Millisecond Time Scale Exchange in Proteins by1H Relaxation Dispersion: An Application to Proteasome Gating Residue Dynamics. J. Am. Chem. Soc.. 132(32):10992-10995. http://dx.doi.org/10.1021/ja104578n
33.
Gans P, Hamelin O, Sounier R, Ayala I, Durá M, Amero C, Noirclerc-Savoye M, Franzetti B, Plevin M, Boisbouvier J. 2010. Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High-Molecular-Weight Proteins. Angewandte Chemie International Edition. 49(11):1958-1962. http://dx.doi.org/10.1002/anie.200905660
34.
Ruschak AM, Velyvis A, Kay LE. 2010. A simple strategy for 13C,1H labeling at the Ile-?2 methyl position in highly deuterated proteins. J Biomol NMR. 48(3):129-135. http://dx.doi.org/10.1007/s10858-010-9449-1
35.
Ayala I, Hamelin O, Amero C, Pessey O, Plevin MJ, Gans P, Boisbouvier J. An optimized isotopic labelling strategy of isoleucine-?2methyl groups for solution NMR studies of high molecular weight proteins. Chem. Commun.. 48(10):1434-1436. http://dx.doi.org/10.1039/c1cc12932e
36.
Isaacson RL, Simpson PJ, Liu M, Cota E, Zhang X, Freemont P, Matthews S. 2007. A New Labeling Method for Methyl Transverse Relaxation-Optimized Spectroscopy NMR Spectra of Alanine Residues. J. Am. Chem. Soc.. 129(50):15428-15429. http://dx.doi.org/10.1021/ja0761784
37.
Ayala I, Sounier R, Usé N, Gans P, Boisbouvier J. 2009. An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR. 43(2):111-119. http://dx.doi.org/10.1007/s10858-008-9294-7
38.
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(51):18340-18350. http://dx.doi.org/10.1021/ja1083656
39.
Fischer M, Kloiber K, Häusler J, Ledolter K, Konrat R, Schmid W. 2007. Synthesis of a13C-Methyl-Group-Labeled Methionine Precursor as a Useful Tool for Simplifying Protein Structural Analysis by NMR Spectroscopy. ChemBioChem. 8(6):610-612. http://dx.doi.org/10.1002/cbic.200600551
40.
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(4):756-769. http://dx.doi.org/10.1016/j.cell.2007.09.039