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Leveraging relaxation-optimized 1H–13CF correlations in 4-19F-phenylalanine as atomic beacons for probing structure and dynamics of large proteins

Nature Chemistry volume 17, pages 835–846 (2025)Cite this article

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Abstract

NMR spectroscopy of biomolecules provides atomic level information into their structure, dynamics and interactions with their binding partners. However, signal attenuation from line broadening caused by fast relaxation and signal overlap often limits the application of NMR to large macromolecular systems. Here we leverage the slow relaxation properties of 13C nuclei attached to 19F in aromatic 19F–13C spin pairs as well as the spin–spin coupling between the fluorinated 13C nucleus and the hydrogen atom at the meta-position to record two-dimensional 1H–13CF correlation spectra with transverse relaxation-optimized spectroscopy selection on 13CF. To accomplish this, we synthesized [4-19F13Cζ; 3,5-2H2ε] Phe, engineered for optimal relaxation properties, and adapted a residue-specific route to incorporate this residue globally into proteins and a site-specific 4-19F Phe encoding strategy. This approach resulted in narrow linewidths for proteins ranging from 30 kDa to 180 kDa, enabling interaction studies with small-molecule ligands without requiring specialized 19F-compatible probes.

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Fig. 1: Synthesis of [4-19F13Cζ; 3,5-2H2ε] Phe and [4-1H13Cζ; 3,5-2H2ε] Phe.
Fig. 2: Ultra-low transverse relaxation rates of [4-19F13Cζ; 3,5-2H2ε] Phe simulated in a 50 kDa protein.
Fig. 3: Narrow linewidths at 50 kDa with the HCF-TROSY.
Fig. 4: 4-19F-Phe-labelled hCAII.
Fig. 5: Deuteration of PARP1 (42 kDa) reduces relaxation of the 4-19F–13C Phe 1Hδ resonances.
Fig. 6: Long-range 1H–13CF-TROSY of deuterated proteasome α7 single-ring particle confirms ultra-slow relaxation of the aromatic 1H–13CF spin system at a molecular mass of 180 kDa.

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Data availability

The density map and the coordinates of the 4-19F Phe hCAII crystal structure were deposited in the Protein Data Bank (PDB) with the following accession code: 7U5W. The search model PDB entry 3S73 is available from the PDB. Pulse sequences and parameter sets are available at the laboratory website (https://artlab.dana-farber.org/downloads). Mass spectrometry data are available at ftp://massive.ucsd.edu/MSV000093932. All other data are available in the paper or in the supplementary materials and as source data. Source data are provided with this paper.

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Acknowledgements

We thank D. Mathieu (Bruker) for help with NMR experiments, G. Whitesides (Harvard University) for kindly providing us the human carbonic anhydrase II plasmid, L. Kay (University of Toronto) for the plasmid of the α7 single ring of the proteasome, and R. Rosenzweig and M. Silva (Weizmann Institute) for the DNAJB1 plasmid and helpful discussions. This work was based on research conducted at the Northeastern Collaborative Access Team beamlines, which were funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on the 24-ID-C beamline was funded by an NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357. S.D.-P. acknowledges funding from the Linde Family Foundation, the Doris Duke Charitable Foundation, Deerfield 3DC and Taiho Pharmaceuticals. V.V. acknowledges funding from the Brazilian National Council for Scientific and Technological Development CNPq grant 200611/2022-4. We acknowledge the generous support of the Austrian Science Fund (Erwin Schrödinger Fellowship FWF3872 awarded to A.B.), grants GM136859 and AI143565 awarded to H.A. and grants from the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number JP22K18374 (to K.T.), the Naito Foundation (to K.T.) and the Takeda Science Foundation (to K.T.). This work was supported in part by a gift from J. Goldberg, Dana-Farber Donor. This work was also supported in part by the GCE4All Biomedical Technology Optimization and Dissemination Center supported by the National Institute of General Medical Science grant RM1-GM144227. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

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Author notes

  1. These authors contributed equally: Denitsa L. Radeva, Sebastian Schindler.

Authors and Affiliations

  1. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Andras Boeszoermenyi, Sebastian Schindler, Veronica Valadares, Krishna M. Padmanabha Das, Abhinav Dubey, Thibault Viennet, Scott Ficarro, Jarred Marto, Ezekiel A. Geffken, Sirano Dhe-Paganon, Hyuk-Soo Seo & Haribabu Arthanari

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

    Andras Boeszoermenyi, Krishna M. Padmanabha Das, Abhinav Dubey, Thibault Viennet, Sirano Dhe-Paganon, Hyuk-Soo Seo & Haribabu Arthanari

  3. Faculty of Chemistry and Pharmacy, Sofia University, Sofia, Bulgaria

    Denitsa L. Radeva, Vladimir M. Gelev, Nikolay Stoyanov, Nikola Burdzhiev & Ognyan Petrov

  4. Department of Chemistry | iNANO, Aarhus University, Aarhus, Denmark

    Thibault Viennet

  5. Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland

    Max Schmitt & Peter Kast

  6. Department of Biochemistry and Biophysics, Oregon State University, GCE4All Research Center, Corvallis, OR, USA

    Nathan D. Alexander, Richard B. Cooley & Ryan A. Mehl

  7. Bruker BioSpin, Fällanden, Switzerland

    Helena Kovacs

  8. Bruker BioSpin, Billerica, MA, USA

    Clemens Anklin

  9. Bruker BioSpin, Rheinstetten, Germany

    Wolfgang Bermel

  10. School of Chemistry and Chemical Engineering, University of Southampton, Southampton, UK

    Ilya Kuprov

  11. Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, Israel

    Ilya Kuprov

  12. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Koh Takeuchi

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  1. Andras Boeszoermenyi
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  2. Denitsa L. Radeva
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  3. Sebastian Schindler
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Contributions

A.B., D.L.R., A.D., V.M.G., S.D.-P., H.-S.S., I.K., M.S., P.K., R.B.C., R.A.M., K.T. and H.A. designed the research; A.B., D.L.R., S.S., V.V., K.M.P.D., A.D., T.V., M.S., P.K., V.M.G., N.S., N.B., O.P., S.F., J.M., E.A.G., S.D.-P., H.-S.S., I.K., N.D.A. and H.A. performed the research; A.B., D.L.R., S.S., N.D.A., M.S., P.K. and V.M.G. generated the reagents; A.B., D.L.R., A.D., T.V., V.M.G., E.A.G., S.D.-P., H.-S.S., H.K., C.A., W.B., I.K., K.T. and H.A. analysed and interpreted the data; A.B., D.L.R., A.D., T.V., V.M.G., S.D.-P., H.-S.S., I.K., K.T. and H.A. wrote the paper with contributions from all other co-authors.

Corresponding authors

Correspondence to Andras Boeszoermenyi, Koh Takeuchi or Haribabu Arthanari.

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V.M.G. is the founder of FB Reagents Ltd., a company that provides isotopically enriched NMR reagents. C.A. works for Bruker BioSpin Corporation, which is a manufacturer of equipment used in this work. W.B. and H.K. were employees at Bruker BioSpin Corporation when this work was conducted and have since retired. H.A. is a co-founder of Quantum Therapeutics Inc., a company that employs computational methods for drug development, although the work presented here does not overlap with that of the company. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 13C-detected HCF-TROSY pulse sequence used to correlate 1Hδ to 19F-attached 13Cζ.

90° pulses are denoted by narrow black rectangles and 180° pulses are denoted by wide black rectangles. Phase cycling and delay times are indicated. A 240 Hz CF coupling is assumed. The CH coupling was set to 20 Hz to maximize magnetization transfer.

Extended Data Fig. 2 The anti-TROSY component of 19F-attached 13C relaxes out of the equation.

(a–c) T2-modulated 1H-13C HSQC spectra of hCAII (29 kDa) at 25°C. The relaxation delays during 13C evolution were set to (a) 0 ms, (b) 8 ms and (c) 12 ms. Dashed red lines connect TROSY and anti-TROSY components along the 13C dimension. A few example peak-pairs were chosen for illustration. (d) Overlay of two HCF-TROSY spectra of hCAII (29 kDa) at 25 °C are shown. One spectrum was recorded with 19F-13C TROSY selection at 600 MHz (blue). The second spectrum was recorded without fluorine selection on a spectrometer not equipped with a fluorine NMR capable probe at 800 MHz (green). The empty space to the left of the spectrum is the space where one would expect anti-TROSY peaks if they were present in the spectrum.

Extended Data Fig. 3 IPAP HCF-TROSY experiment on small proteins.

IPAP1 (a) and IPAP2 (b) spectra recorded with 90-degree phase offset on 13C. (c) Addition of the two spectra removes the broadened anti-TROSY signals. (d) 2D HETCOR pulse sequence with a 180-degree 19F pulse and IPAP phase cycle.

Extended Data Fig. 4 TROSY selection for [4-1H13Cζ; 3,5,-2H2ε] Phe-labelled hCAII.

(a) Overlay of four out-and-stay 1H-13C correlation spectra recorded on hCAII at 25°C and 800 MHz where TROSY and anti-TROSY components of a coupled 1H-13C spin system were recorded with spin-state selection. TROSY components with respect to 13C are to the left (blue and green peaks) and anti-TROSY components are to the right (pink and brown peaks). (b) Out-and-stay 1H-13C correlation spectra that select for the top left (TROSY) component at 25 °C (blue) and 5 °C (purple), respectively.

Extended Data Fig. 5 Optimization of protein labelling with 4-19F Phe.

(a) 19F-NMR spectra of 900 μM GB1 samples at 25 °C produced with varying concentrations of 4-19F Phe in the growth medium. (b) Labelling efficiency of GB1, measured by mass spectrometry (MS), as a function of 4-19F Phe concentration with and without the co-transformed plasmids pHE3-M4G or pHE3-W. (c) MS-derived labelling efficiencies for DNAJB1 (DNAJ), the proteasome α7 single-ring (proteasome) and Keap1 ΔN-term.

Extended Data Fig. 6 Replacing protons within 5 Å of the 4-19F Phe 1Hδ protons reduces relaxation 4-19F Phe residues in hCAII.

(a) Structure of hCAII with 4-19F Phe residues highlighted. The Zn(II) ion close to the active center is shown in pink. (b) Theoretical calculation of the relaxation rates of the 4-19F Phe 1Hδ protons in hCAII, assuming a correlation time of 30 ns. In the first scenario all nearby hydrogen atoms are assumed to be protons (blue) and in the second scenario all nearby hydrogen atoms are assumed to be deuterons (green). (c) Theoretical calculations simulating the effect of deuteration on 1Hδ transverse relaxation in hCAII at 800 MHz and with 30 ns correlation time plotted for individual 1Hδ resonances (blue). All surrounding hydrogen atoms are treated as protons, except for the 4-19F Phe Hε atoms, which are treated as deuterons. (Red) All surrounding hydrogen atoms are treated as deuterons, except for the 4-19F Phe Hβ atoms, which are treated as protons. (Orange) All hydrogen atoms are treated as deuterons. (d) Theoretical calculations simulating a correlation time of 85 ns for hCAII, to mimic the estimated correlation time of the 180-kDa α7 single-ring of the 20S proteasome CP at 35 °C. All other parameters are as described in (c).

Extended Data Fig. 7 Delayed decoupling enhances sensitivity of HCF-TROSY.

(a) Reference experiment and (b) delayed decoupling HCF-TROSY. (c) Relative peak intensities measured in the delayed decoupling HCF-TROSY (PARP1deut DD, hot pink filled circles), and the corresponding reference experiment (PARP1deut noDD, light pink filled squares) are plotted.

Source data

Extended Data Fig. 8 19F-13C and HCF-TROSY reveal Olaparib binding to PARP1.

(a) 19F-13C TROSYs of 400 μM protonated [4-19F13Cζ; 3,5-2H2ε] Phe-labelled PARP1 in presence (navy) and absence (pink) of 400 μM Olaparib. (b) HCF-TROSYs of the samples shown in (a) following the same colour code. One small negative peak (noise) is shown in grey.

Extended Data Fig. 9 Site-specifically incorporated [4-19F13Cζ; 3,5-2H2ε] Phe in GFP provides sharp cross-peaks.

(a) 19F-13C TROSY and (b) HCF-TROSY of site-specifically labelled [4-19F13Cζ; 3,5-2H2ε] Phe in GFP recorded at 25 °C.

Extended Data Fig. 10

19F-substituted aromatic amino acids with three-bond coupling between 19F-13C and 1H.

Supplementary information

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Supplementary Methods, Figs. 1–5, Tables 1 and 2, and Notes 1 and 2.

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Source data

Source Data Fig. 3

Source data for Fig. 3.

Source Data Fig. 6

Source data for Fig. 6.

Source Data Extended Data Fig./Table 7

Source data for Extended Data Fig. 7.

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Boeszoermenyi, A., Radeva, D.L., Schindler, S. et al. Leveraging relaxation-optimized 1H–13CF correlations in 4-19F-phenylalanine as atomic beacons for probing structure and dynamics of large proteins. Nat. Chem. 17, 835–846 (2025). https://doi.org/10.1038/s41557-025-01818-8

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