Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

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Pulse sequences and parameter sets are available as Supplementary Software and at the laboratory website, https://artlab.dana-farber.org/19f_13c_aromatictrosy.html.

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We thank K.E. Leigh and M. Kostic for help in proofreading of the manuscript presented here and discussions regarding the work, and D. Baldisseri for help with the NMR experiments. We are especially grateful to L.E. Kay (Departments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, Ontario, Canada) for sharing the plasmid carrying the single-ring α7 proteasome particle with us. This research was supported by the NIH (grant nos. GM047467, GM129026 and AI037581 to G.W.), the Claudia Adams Barr Program for Innovative Cancer Research (H.A.), the Austrian Science Fund FWF (Schrödinger Fellowship no. J3872-B21 to A.B.), and the National Health and Medical Research Council Australia (C. J. Martin Fellowship no. APP1090444 to S.C.). Maintenance of the NMR instruments used for this research was supported by NIH grant no. EB002026.

Author information

Author notes

  1. These authors contributed equally: Andras Boeszoermenyi, Sandeep Chhabra.


  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    • Andras Boeszoermenyi
    • , Sandeep Chhabra
    • , Abhinav Dubey
    • , Meng Zhang
    • , Gerhard Wagner
    •  & Haribabu Arthanari
  2. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Andras Boeszoermenyi
    • , Sandeep Chhabra
    • , Abhinav Dubey
    •  & Haribabu Arthanari
  3. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    • Sandeep Chhabra
  4. Faculty of Chemistry and Pharmacy, Sofia University, Sofia, Bulgaria

    • Denitsa L. Radeva
    • , Nikola T. Burdzhiev
    • , Christo D. Chanev
    • , Ognyan I. Petrov
    •  & Vladimir M. Gelev
  5. Bruker Biospin, Billerica, MA, USA

    • Clemens Anklin
  6. Bruker Biospin, Fällanden, Switzerland

    • Helena Kovacs
  7. School of Chemistry, University of Southampton, Highfield, Southampton, UK

    • Ilya Kuprov
  8. Molecular Profiling Research Center for Drug Discovery , National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

    • Koh Takeuchi


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A.B., S.C., G.W., V.M.G., I.K., K.T., and H.A. designed the research. A.B., S.C., A.D., D.L.R., N.T.B., C.D.C., O.I.P., V.M.G., C.A., M.Z., H.K., I.K., K.T., and H.A. performed experiments. A.B., S.C., A.D., V.M.G., H.K., I.K., and H.A. analyzed the data. A.B., S.C., A.D., G.W., I.K., D.L.R., V.M.G., K.T., and H.A. wrote the paper.

Competing interests

V.M.G. is the founder of FB Reagents Ltd, a company that provides isotopically enriched NMR reagents. C.A. and H.K. work for Bruker Biospin Corporation, which is a manufacturer of equipment used in this work.

Corresponding authors

Correspondence to Koh Takeuchi or Haribabu Arthanari.

Integrated supplementary information

  1. Supplementary Figure 1 Calculated magnetic field dependence of the transverse relaxation rates for 13CF nuclei and 19FC nuclei in aromatic amino acids with various 19F substitutions.

    Blue triangles represent the relaxation rates of decoupled resonances, and red squares represent those of TROSY resonances. The points are connected by a dashed line to provide visual clarity. The relaxation rates are calculated for the respective 19F-bearing amino acids incorporated into a protein with a rotational correlation time (τc) of 25 ns.

  2. Supplementary Figure 2 Selective detection of the TROSY component.

    a,b, 13C-19F HSQCs of 3-19F13C Tyr-labeled MBP recorded at 600 MHz: (a) decoupled and (b) coupled. The expected narrowing for the TROSY component is observed in b. A dashed rectangle highlights the four components of one decoupled spin-pair (red). The top right component is clearly more intense than the top left component and the bottom components, which are anti-TROSY with respect to 13CF are substantially broadened. In addition, two intense (more mobile) 13CF TROSY peaks are highlighted and a 1D trace identifies the component to the right (top right) as the TROSY component. c, The TROSY component was selectively observed by the ST2PT scheme. The negative signal (purple) originates from imperfect cancelation of the anti-TROSY component.

  3. Supplementary Figure 3 Sensitivity-enhanced (SE) and non-SE out-and-back 13C-19F TROSYs exhibit comparable sensitivity.

    a,b, Pulse schemes for (a) SE and (b) non-SE TROSY versions. c,d, The TROSY spectra of 3-19F13C Tyr-labeled MBP recorded at 600 MHz with SE (c) and non-SE (d) pulse sequences show no substantial difference in sensitivity. Owing to the fast-relaxing 19F magnetization spending more time in the transverse plane in the SE version of the pulse sequence, the typical sensitivity gains associated with the SE experiment were offset. All narrow rectangles denote 90° pulses, and broad rectangles represent 180° pulses. 1H refocusing and decoupling were not possible because of the architecture of the probe, but could in principle be implemented and hence are represented in the pulse sequence. The duration of the delay T was set to 2.08 ms (= 1/2 JCF). Δ is a delay to refocus any chemical shift evolution occurring during evolution of the first point in the indirect dimension, whereas Δ1 refocuses chemical shift evolution occurring during the last gradient and the gradient recovery period. All pulses, unless otherwise noted, are applied along the x-axis. The phase cycles used are indicated under the respective pulse programs. The quadrature detection in the indirect dimension for a was achieved using the Echo-Anti-echo detection scheme, and for b it was done using the States-TPPI approach by incrementing the phase ϕ1. The duration and strengths of the gradients are g0 = (the length of the indirect encoding period, 1.65 Gauss/cm); g1 = (750 μs, 16.5 Gauss/cm); g2 = (500 μs, 44 Gauss/cm); g3 = (750 μs, 24.75 Gauss/cm); g4 = (750 μs, 27.5 Gauss/cm) and g5 = (500 μs, 23.51 Gauss/cm) in the SE version of the experiment (a). The duration and strengths of the gradients in the non-SE TROSY experiments (b) were g0 = (the length of the indirect encoding period, 1.65 Gauss/cm); g1 = (700 ms, 12.9 Gauss/cm); g2 = (500 ms, –33.0 Gauss/cm); g3 = (700 ms, 22 Gauss/cm); g4 = (700 ms, 31.68 Gauss/cm) and g5 = (500 ms, 23.52 Gauss/cm).

  4. Supplementary Figure 4 Contribution of DD and CSA mechanisms to the relaxation rates of various product states in the 13C-19F TROSY experiment.

    Relaxation rates of 13C-19F product states are shown, with contributions from 19F-13C DD (green), 19F CSA (red), and 13C CSA (blue). The theoretical calculations were performed for 3-19F13C Tyr incorporated into a protein with a rotational correlation time (τc) of 25 ns at a magnetic field strength of 600 MHz.

  5. Supplementary Figure 5 Pulse schemes for 13C-start, 19F-detected TROSY and 19F-start, 13C-detected TROSY.

    Narrow rectangular bars denote flip angles of 90°, and wide bars denote flip angles of 180°. 1H refocusing and decoupling during acquisition were not possible because of the architecture of the probe, but could in principle be implemented and hence are represented in the pulse sequence. The duration of the delay T was set to 2.08 ms (= 1/2 JCF). All pulses, unless otherwise noted, are applied along the x-axis. The phase cycles used are indicated under the respective pulse programs. The indirect dimension is acquired using the Echo-Anti-echo detection scheme by changing ϕ1 for quadrature detection. For both the 13C-start, 19F-detected TROSY and 19F-start, 13C-detected TROSY, ϕ2 and ϕ3 are set to y and –y, respectively. The duration and strengths of the gradients are g3 = (750 μs, 24.75 Gauss/cm) and g4 = (750 μs, 27.5 Gauss/cm).

  6. Supplementary Figure 6 Illustration of spin-state selection by ST2PT scheme.

    Overlay of four out-and-stay 19F-13C correlation spectra where the various components of a coupled 19F-13C spin system were recorded with spin-state selection on an MBP sample at 600 MHz, labeled with 3-19F13C Tyr. The four components corresponding to a single 19F-13C correlation (a representative 3-19F13C Tyr residue in MBP) are highlighted in a dashed rectangular box. The top-right component (brown) is the TROSY resonance.

  7. Supplementary Figure 7 Longitudinal relaxation times (T1) for 13CF and 19FC nuclei in 3-19FC Tyr-labeled GB1 at 500 MHz.

    a,b, 1D 13CF (a) and 1D 19FC (b) spectra depicting the region with peaks from 3-19F13C Tyr. c,d, Longitudinal relaxation curves (blue) and relaxation times (T1) fitted to experimentally measured values (red points) for peaks 1–6 in a (c) and b (d).

  8. Supplementary Figure 8 Calculated transverse relaxation rates (R2) as a function of rotational correlation time.

    ad, R2 for (a) 13CF and (c) 19FC in 3-19F13C Tyr and (b) 13CH and (d) 1HC in Tyr as a function of rotational correlation times (τc) ranging from 5 to 95 ns at a field strength of 600 MHz. Blue triangles represent the R2 of decoupled resonances, and red squares represent those of the narrow TROSY resonances. The points are connected by a dashed line to provide visual clarity.

  9. Supplementary Figure 9 19F-13C TROSY enables 13C-detection with high resolution for large-molecular-weight systems: comparing the TROSY and anti-TROSY components.

    a,b, The TROSY cross-peaks in a at 25 °C (e.g., orange peak) are considerably sharper than their anti-TROSY counterparts (e.g., blue peak) in b. The bottom panels show spectra recorded at 10 °C, where the temperature was decreased to mimic the relaxation of a larger-molecular-weight system. c,d, TROSY cross-peaks in c remain sharp and intense (e.g., red peak), whereas in d the corresponding anti-TROSY cross-peaks broaden considerably and are barely visible (e.g., dark blue peak).

  10. Supplementary Figure 10 High-resolution 19F-13C TROSY facilitates the detection of minor conformations.

    Low-population cross-peaks in the 19F-13C TROSY spectrum of MBP are shown in green circles. These resonances are likely to have originated from rotamers of 3-19F13C Tyr due to (1) rotation along the Cγ–Cβ bond or (2) rotation along the phenolic Cζ–O bond, which determines the proximity of the hydroxyl hydrogen atom to the fluorine atom. Similarly, slow flipping of the hydroxyl group around the phenolic Cζ–O bond between an intra-residue O-H-F and an inter-residue H-bonding configuration can produce two sets of resonances of unequal intensity. Such rotamers of 3-19F Tyr have been previously observed by X-ray crystallography (J. Mol. Biol. 281, 323–339; 1998). In a few instances, for the very strong cross-peaks, we observed a minor conformation that corresponds to the non-TROSY component along the 13C dimension; this is marked by a red solid rectangle. The non-TROSY component can be recognized by its 19F resonance frequency, which is identical to that of its TROSY counterpart with a 13C frequency separation of ~240 Hz, corresponding to the 1JFC coupling constant.

  11. Supplementary Figure 11 Calculated magnetic field dependence of R2 for 13CF and 19FC nuclei in various 19F-substituted nucleobases.

    Blue triangles represent the R2 of decoupled resonances, and red squares represent those of TROSY resonances. The relaxation rates are calculated for the respective 19FC-bearing nucleobase assumed to be incorporated into a nucleic acid with a rotational correlation time (τc) of 25 ns. The points are connected by a dashed line to provide visual clarity.

  12. Supplementary Figure 12 R2 calculated for 13CF, 13CH, 19FC, and 1HC nuclei in 5-19F uracil or uracil as a function of magnetic field strength.

    a,b, R2 of 13CF as part of 5-19F uracil (a) and R2 of 5-13CH in uracil (b). c,d, R2 of the uracil 5-19FC uracil (c) and R2 of 5-1HC (d). Here uracil and 5-19F uracil relaxation times are calculated with a rotational correlation time (τc) of 25 ns. Red squares represent the R2 of the TROSY components, and blue triangles represent the R2 of the decoupled resonances. The points are connected by a dashed line to provide visual clarity.

  13. Supplementary Figure 13 1H decoupling improves resolution and sensitivity of the 19F-13C TROSY experiment.

    a, 13C-19F TROSY-SE of 3-19F13C Tyr-labeled GB1 without 1H decoupling. Here, 3JCH coupled peaks are further split by the 2JCC into a doublet of doublets along the 13C dimension. This is further emphasized by the vertical 1D trace at –137 p.p.m. on the 19F axis. b, 13C-19F TROSY-SE spectrum of GB1 with 1H decoupling. The 1H coupling is removed, and the peaks are pure doublets in the 13C dimension, owing to the ~7-Hz 2JCC splitting.

  14. Supplementary Figure 14 The 13CF TROSY resonance is the slowest relaxing nucleus in biological NMR.

    a,b, Theoretical transverse relaxation rates of 13CF, 15NH and 13CH at the magnetic field strength where the TROSY effect for any given spin-pair is maximized, calculated for (a) a 42-kDa protein (τc at 25 °C = 25 ns) and (b) a 110-kDa protein (τc at 25 °C = 65 ns). Simulated free induction decays (top) compare and visualize the influence of transverse relaxation on the height and line width of the TROSY resonance of the aromatic 13CF, aromatic 13CH and backbone 15NH spin states.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figs. 1–14, Supplementary Table 1, and Supplementary Notes 1–3

  2. Reporting Summary

  3. Supplementary Software

    Pulse programs and parameter sets.

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