13CHD2–CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins

Abstract

An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of 13CHD2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360 kDa half-proteasome, establish that the sensitivity of this 13CHD2 13C–CEST technique can be upwards of a factor of 5 times higher than for a previously published 13CH3 13C–CEST approach (Bouvignies and Kay in J Biomol NMR 53:303–310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins.

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References

  1. Abragam A (1961) Principles of nuclear magnetism. Clarendon Press, Oxford

    Google Scholar 

  2. Allard P, Helgstrand M, Hard T (1998) The complete homogeneous master equation for a heteronuclear two-spin system in the basis of cartesian product operators. J Magn Reson 134:7–16

    Article  ADS  Google Scholar 

  3. 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 

  4. Bouvignies G, Kay LE (2012) A 2D (1)(3)C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding. J Biomol NMR 53:303–310

    Article  Google Scholar 

  5. Bouvignies G, Vallurupalli P, Kay LE (2014) Visualizing side chains of invisible protein conformers by solution NMR. J Mol Biol 426:763–774

    Article  Google Scholar 

  6. Clore GM, Gronenborn AM (1991) Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy. Science 252:1390–1399

    Article  ADS  Google Scholar 

  7. Fawzi NL, Ying J, Ghirlando R, Torchia DA, Clore GM (2011) Atomic-resolution dynamics on the surface of amyloid-beta protofibrils probed by solution NMR. Nature 480:268–272

    Article  ADS  Google Scholar 

  8. Forsen S, Hoffman RA (1963) Study of moderately rapid chemical exchange reactions by means of nuclear magnetic double resonance. J Chem Phys 39:2892–2901

    Article  ADS  Google Scholar 

  9. Gans P et al (2010) Stereospecific isotopic labeling of methyl groups for NMR spectroscopic studies of high molecular weight proteins. Angew Chem Int Ed 49:1958–1962

    Article  Google Scholar 

  10. Gardner KH, Kay LE (1997) Production and incorporation of 15N, 13C, 2H (1H–δ1 methyl) isoleucine into proteins for multidimensional NMR studies. J Am Chem Soc 119:7599–7600

  11. Gelis I et al (2007) Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131:756–769

    Article  Google Scholar 

  12. 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 

  13. Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-,13C-,2H-labeled proteins. J Biomol NMR 13:369–374

    Article  Google Scholar 

  14. Grzesiek S, Anglister J, Ren H, Bax A (1993) 13C line narrowing by 2H decoupling in 2/13C/15N-enriched proteins. Applications to triple resonance 4D J-connectivity of sequential amides. J Am Chem Soc 115:4369–4370

    Article  Google Scholar 

  15. Guenneugues M, Berthault P, Desvaux H (1999) A method for determining B1 field inhomogeneity. Are the biases assumed in heteronuclear relaxation experiments usually underestimated? J Magn Reson 136:118–126

    Article  ADS  Google Scholar 

  16. Helgstrand M, Hard T, Allard P (2000) Simulations of NMR pulse sequences during equilibrium and non- equilibrium chemical exchange. J Biomol NMR 18:49–63

    Article  Google Scholar 

  17. Huth JR, Bewley CA, Jackson BM, Hinnebusch AG, Clore GM, Gronenborn AM (1997) Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR. Protein Sci 6:2359–2364

    Article  Google Scholar 

  18. Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Appl Calmodulin Biochem 29:4659–4667

    Google Scholar 

  19. 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 129:15428–15429

    Article  Google Scholar 

  20. Ishima R, Louis JM, Torchia DA (1999) Transverse C-13 relaxation of CHD2 methyl isotopomers to detect slow conformational changes of protein side chains. J Am Chem Soc 121:11589–11590

    Article  Google Scholar 

  21. Kainosho M, Torizawa T, Iwashita Y, Terauchi T, Ono AM, Guntert P (2006) Optimal isotope labelling for NMR protein structure determinations. Nature 440:52–57

    Article  ADS  Google Scholar 

  22. Kay LE, Torchia DA (1991) The effects of dipolar cross-correlation on 13C methyl-carbon T1, T2 and NOE measurements in macromolecules. J Magn Reson 95:536–547

    ADS  Google Scholar 

  23. 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

    ADS  Google Scholar 

  24. 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 

  25. Korzhnev DM, Religa TL, Banachewicz W, Fersht AR, Kay LE (2010) A transient and low-populated protein-folding intermediate at atomic resolution Science 329:1312–1316

    Google Scholar 

  26. Levitt MH (1982) Symmetrical composite pulse sequences for NMR population-inversion. 2. Compensation of resonance offset. J Magn Reson 50:95–110

    ADS  Google Scholar 

  27. Lipari G, Szabo A (1982) Model-free approach to the interpretation of nuclear magnetic relaxation in macromolecules: 2. Analysis of experimental results. J Am Chem Soc 104:4559–4570

    Article  Google Scholar 

  28. Lowe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R (1995) Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 268:533–539

    Article  ADS  Google Scholar 

  29. McConnell HM (1958) Reaction rates by nuclear magnetic resonance. J Chem Phys 28:430–431

    Article  ADS  Google Scholar 

  30. Messerlie BA, Wider W, Otting G, Weber C, Wuthrich K (1989) J Magn Reson 85:608–612

    ADS  Google Scholar 

  31. Nikonowicz EP, Sirr A, Legault P, Jucker FM, Baer LM, Pardi A (1992) Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucl Acids Res 20:4507–4513

    Article  Google Scholar 

  32. Ollerenshaw JE, Tugarinov V, Skrynnikov NR, Kay LE (2005) Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins. J Biomol NMR 33:25–41

    Article  Google Scholar 

  33. Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy. J Magn Reson 93:151–170

    ADS  Google Scholar 

  34. Press WH, Flannery BP, Teukolsky SA, Vetterling WT (1988) Numerical recipes in C. Cambridge University Press, Cambridge

    MATH  Google Scholar 

  35. Religa TL, Kay LE (2010) Optimal methyl labeling for studies of supra-molecular systems. J Biomol NMR 47:163–169

    Article  Google Scholar 

  36. Religa TL, Ruschak AM, Rosenzweig R, Kay LE (2011) Site-directed methyl group labeling as an NMR probe of structure and dynamics in supramolecular protein systems: applications to the proteasome and to the ClpP protease. J Am Chem Soc 133:9063–9068

    Article  Google Scholar 

  37. Rosenzweig R, Kay LE (2014) Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu Rev Biochem 83:291–315

    Article  Google Scholar 

  38. Ruschak AM, Kay LE (2009) Methyl groups as probes of supra-molecular structure, dynamics and function. J Biomol NMR 46:75–87

    Article  Google Scholar 

  39. Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog Nucl Magn Reson Spectrosc 34:93–158

    Article  Google Scholar 

  40. 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 

  41. Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broadband decoupling: WALTZ-16. J Magn Reson 52:335–338

    ADS  Google Scholar 

  42. Shaka AJ, Lee CJ, Pines A (1988) Iterative schemes for bilinear operators—application to spin decoupling. J Magn Reson 77:274–293

    ADS  Google Scholar 

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

    Article  Google Scholar 

  44. Tugarinov V, Kay LE (2004) An isotope labeling strategy for methyl TROSY spectroscopy. J Biomol NMR 28:165–172

    Article  Google Scholar 

  45. Tugarinov V, Kay LE (2005a) Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. ChemBioChem 6:1567–1577

    Article  Google Scholar 

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

    Article  Google Scholar 

  47. Tugarinov V, Hwang P, Ollerenshaw J, 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 

  48. Vallurupalli P, Kay LE (2013) Probing slow chemical exchange at carbonyl sites in proteins by chemical exchange saturation transfer NMR spectroscopy. Angew Chem Int Ed Engl 52:4156–4159

    Article  Google Scholar 

  49. Vallurupalli P, Bouvignies G, Kay LE (2012) Studying ‘invisible’ excited protein states in slow exchange with a major conformation. J Am Chem Soc 134:8148–8161

    Article  Google Scholar 

  50. 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:e43725. doi:10.1371/journal.pone.0043725

    Article  ADS  Google Scholar 

  51. Wang AC, Bax A (1993) Minimizing the effects of radio-frequency heating in multidimensional NMR experiments. J Biomol NMR 3:715–720

    Article  Google Scholar 

  52. Werbelow LG, Grant DM (1977) Intramolecular dipolar relaxation in multispin systems. Adv Magn Reson 9:189–299

    Article  Google Scholar 

  53. Werbelow LG, Marshall AG (1973) Internal rotation and nonexponential methyl nuclear relaxation for macromolecules. J Magn Reson 11:299–313

    ADS  Google Scholar 

  54. Yamazaki T, Lee W, Arrowsmith CH, Muhandiram DR, Kay LE (1994) A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity. J Am Chem Soc 116:11655–11666

    Article  Google Scholar 

  55. Zhou JY, van Zijl PCM (2006) Chemical exchange saturation transfer imaging and spectroscopy. Prog Nucl Magn Reson Spectrosc 48:109–136

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. L.E.K holds a Canada Research Chair in Biochemistry.

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Correspondence to Lewis E. Kay.

Appendix: Effects of 2H spin relaxation on 13CHD2–CEST profiles

Appendix: Effects of 2H spin relaxation on 13CHD2–CEST profiles

In a previous set of papers we have discussed the effects of homonuclear scalar couplings on CEST profiles and presented a simple approach for data analysis (Bouvignies et al. 2014; Vallurupalli and Kay 2013). By means of example, consider the case where 13CO CEST profiles are obtained from measurements on a uniformly 13C labeled protein sample. Each 13CO dip is split into a doublet by the approximately 50 Hz 13CO–13Cα scalar coupling (JCαCO). Although multiplet components are generally not observed in 13CO CEST profiles when weak B1 fields on the order of 20 Hz or greater are used, the resultant dips are nevertheless broadened by the unresolved couplings and this effect should be taken into account in fits of the profiles to extract accurate exchange parameters. This can be most easily accomplished by solving the Bloch–McConnell equations (McConnell 1958) for two 13CO lines, separated by JCαCO, that correspond to 13Cα spins in the up/down positions. Here it is assumed that the longitudinal relaxation of 13Cα is slow compared to the rate of exchange between conformers so that the resulting CEST profiles are the simple sum of a pair of profiles, one for 13Cα spin up and a second for 13Cα spin down. This procedure can be generalized when more than a single homonuclear coupling is present, as described previously (Bouvignies et al. 2014). The situation is more complex for 13CHD2–CEST when 2H decoupling is not used. In this case each 13C dip is split into a 1:2:3:2:1 pentet structure from 13C–2H scalar coupling interactions. However, the 2H spin flip rate cannot be assumed to be slow compared to the conformational exchange process, so that averaging of the multiplet components can occur simultaneously with chemical exchange, necessitating a more complex treatment. In what follows we assume that 1H decoupling is employed during the CEST interval, as indicated in the pulse scheme of Fig. 3, and thus neglect the influence of the one bond 1H–13C scalar coupling.

The effects of 2H spin flips on 13CHD2 CEST profiles are best considered by first separating the methyl 13C j magnetization \( j \in \{ x,y,z\} \) into distinct components according to the spin states of the pair of coupled deuterons. In what follows we consider initially a basis comprised of 6 operators, L1 j L6 j , defined as,

$$ \begin{aligned} L1_{j} & = C_{j} [(1,1)] = 0.25C_{j} (D_{z,1}^{2} + D_{z,1} )(D_{z,2}^{2} + D_{z,2} ) \\ L2_{j} & = C_{j} [(1,0) + (0,1)] = 0.5C_{j} \{ (D_{z,1}^{2} + D_{z,1} )(1 - D_{z,2}^{2} ) + (1 - D_{z,1}^{2} )(D_{z,2}^{2} + D_{z,2} )\} \\ L3_{j} & = C_{j} [(0,0)] = C_{j} \{ (1 - D_{z,1}^{2} )(1 - D_{z,2}^{2} )\} \\ L4_{j} & = C_{j} [(1, - 1) + ( - 1,1)] = 0.25C_{j} \{ (D_{z,1}^{2} + D_{z,1} )(D_{z,2}^{2} - D_{z,2} ) + (D_{z,1}^{2} - D_{z,1} )(D_{z,2}^{2} + D_{z,2} )\} \\ L5_{j} & = C_{j} [( - 1,0) + (0, - 1)] = 0.5C_{j} \{ (D_{z,1}^{2} - D_{z,1} )(1 - D_{z,2}^{2} ) + (1 - D_{z,1}^{2} )(D_{z,2}^{2} - D_{z,2} )\} \\ L6_{j} & = C_{j} [( - 1, - 1)] = 0.25C_{j} (D_{z,1}^{2} - D_{z,1} )(D_{z,2}^{2} - D_{z,2} ). \\ \end{aligned} $$
(7)

In Eq. (7) C j [(A, B)] denotes the j component of 13C magnetization coupled to 2H spin 1 with magnetic quantum number A, A \( \in \) (−1, 0, 1) and 2H spin 2 with magnetic quantum number B, and D z,k is the z-component of magnetization from 2H spin k \( \in \) (1, 2). A straightforward, albeit lengthy, calculation shows that the relaxation of L1 j L6 j , considering 2H contributions and adding 13C relaxation (\( R_{1}^{C} \) or \( R_{2}^{C} \)) in an ad hoc manner, is given by

$$ \frac{{d\vec{L}_{j} }}{dt} = - \tilde{R}{}_{j}\vec{L}{}_{j} $$
(8)

where

$$ \vec{L}_{j} = \{ L1_{j} ,L2_{j} ,L3_{j} ,L4_{j} ,L5_{j} ,L6_{j} \}^{T} $$
(9)

and

$$ \tilde{R}_{j} = \left( {\begin{array}{*{20}c} { {- 2\kappa_{1}} {-2\kappa_{2}} {-R_{p}^{C} }} & {\kappa_{1} } & 0 & {\kappa_{2} } & 0 & 0 \\ {2\kappa_{1} } & { {-3\kappa_{1}} {-\kappa_{2}} {-R_{p}^{C} }} & {2\kappa_{1} } & {\kappa_{1} } & {\kappa_{2} } & 0 \\ 0 & {\kappa_{1} } & { {-4\kappa_{1}} {-R_{p}^{C}} } & 0 & {\kappa_{1} } & 0 \\ {2\kappa_{2} } & {\kappa_{1} } & 0 & { {-2\kappa_{1}} {-2\kappa_{2}} {-R_{p}^{C}} } & {\kappa_{1} } & {2\kappa_{2} } \\ 0 & {\kappa_{2} } & {2\kappa_{1} } & {\kappa_{1} } & { {-3\kappa_{1}} {-\kappa_{2}} {-R_{p}^{C}} } & {2\kappa_{1} } \\ 0 & 0 & 0 & {\kappa_{2} } & {\kappa_{1} } & { {-2\kappa_{1}} {-2\kappa_{2}} {-R_{p}^{C} } }\\ \end{array} } \right) $$
(10)

where the superscript T in Eq. (9) is the transpose operator,

$$ \begin{aligned} \kappa_{1} & = \frac{6}{5}c^{2} J(\omega_{D} ) \\ \kappa_{2} & = \frac{12}{5}c^{2} J(2\omega_{D} ), \\ \end{aligned} $$
(11)

\( \frac{2c}{\pi } \sim 165\;{\text{kHz}} \) is the quadrupolar coupling constant and J(ω D ) is given by Eq. (2) of the main text. In Eq. (10) the value of p is 1 if j = z (longitudinal relaxation) or 2 if \( j \in \{ x,y\} \) (transverse relaxation).

Eq. (8) is written in a basis where \( j \in \{ x,y,z\} \). The above equations can be ‘expanded’ by explicitly including terms for each of the x, y and z components so that the 6 × 6 \( \tilde{R} \) matrix above (\( \tilde{R}_{6} \)) becomes an 18 × 18 matrix, \( \tilde{R}_{18} = \tilde{I}_{3} \otimes \tilde{R}_{6} \) where \( \tilde{I}_{3} \) is a 3 × 3 identity matrix and \( \vec{L} = \{ L1_{x} ,L2_{x} ,L3_{x} ,L4_{x} ,L5_{x} ,L6_{x} , \ldots ,L6_{z} \}^{T} \). The effects of chemical shift and 2H–13C scalar-coupled evolution couple x and y components of magnetization and are included into matrix \( \tilde{R}_{18} \) by noting that

$$ \begin{aligned} \frac{{dC_{x} [(A,B)]}}{dt} & = - (\omega + (A + B)2\pi J_{CD} )C_{y} [(A,B)] \\ \frac{{dC_{y} [(A,B)]}}{dt} & = (\omega + (A + B)2\pi J_{CD} )C_{x} [(A,B)] \\ \end{aligned} $$
(12)

Finally, two-site chemical exchange, \( G\mathop{\mathop{\rightleftarrows}\limits_{k_{EG}}}\limits^{k_{GE}}E \), is taken into account (Allard et al. 1998; Helgstrand et al. 2000) by a further expansion of the equations with \( \vec{L} = \{ L1_{x}^{G} ,L2_{x}^{G} ,L3_{x}^{G} ,L4_{x}^{G} ,L5_{x}^{G} ,L6_{x}^{G} , \ldots ,L6_{z}^{G} ,L1_{x}^{E} \ldots ,L6_{z}^{E} \}^{T} \)

$$ \tilde{R^{\prime}}_{36} = \left( {\begin{array}{*{20}c} {\tilde{R}_{18}^{G} } & {\tilde{0}_{18} } \\ {\tilde{0}_{18} } & {\tilde{R}_{18}^{E} } \\ \end{array} } \right) + \left( {\begin{array}{*{20}c} { - k_{GE} } & {k_{EG} } \\ {k_{GE} } & { - k_{EG} } \\ \end{array} } \right) \otimes \tilde{I}_{18} $$
(13)

where the superscripts G and E denote the ground and excited states and \( \tilde{O}_{18} \) is an 18 dimensional null matrix. It is assumed that R C1 values are identical in \( \tilde{R}_{18}^{G} \) and \( \tilde{R}_{18}^{E} \) but that corresponding spins in ground and excited states have distinct R C2 rates. Software for fitting exchange data is available upon request.

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Rennella, E., Huang, R., Velyvis, A. et al. 13CHD2–CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins. J Biomol NMR 63, 187–199 (2015). https://doi.org/10.1007/s10858-015-9974-z

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Keywords

  • CEST
  • Methyl-labeling
  • 13CHD2
  • Chemical exchange
  • Proteasome
  • Sensitivity enhancement