the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 24 Feb 2026
Accelerated 19F biomolecular magic-angle spinning NMR with paramagnetic dopants
Abstract. The advantageous characteristics attributed to the 19F nucleus have made it a popular target for NMR once again in recent years. Aside from solution NMR, an increasing number of studies have been conducted applying solid-state magic-angle-spinning NMR to fluorine-labeled samples. Here, the high chemical shift anisotropy and strong dipolar couplings can be utilized to get structural insights into proteins and measure long distances. Despite increasing popularity and promising benefits, the sensitivity of biomolecular 19F MAS NMR often suffers from slow longitudinal T1 relaxation and therefore long recycle delays. In this work, we expand paramagnetic doping, an approach commonly used to reduce proton T1 relaxation times, to 19F-labeled biological samples. We study the effect of Gd(DTPA) and Gd(DTPA-BMA) on 19F and 13C T1 and T2 relaxation in a [5-19F13C]-tryptophan-labeled protein via 19F-detected MAS NMR experiments. The observed paramagnetic relaxation enhancement substantially reduces measurement times of 19F MAS NMR experiments without compromising resolution. Additionally, we report the chemical-shift assignments of all four fluorotryptophan signals in the 12 × 39 kDa large protein using a mutagenesis approach.
Competing interests: At least one of the (co-)authors is a member of the editorial board of Magnetic Resonance.
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, published maps, institutional affiliations, or any other geographical representation in this paper. While Copernicus Publications makes every effort to include appropriate place names, the final responsibility lies with the authors. Views expressed in the text are those of the authors and do not necessarily reflect the views of the publisher.- Preprint
(2332 KB) - Metadata XML
-
Supplement
(391 KB) - BibTeX
- EndNote
Status: open (until 24 Mar 2026)
- RC1: 'Comment on mr-2026-3', Gottfried Otting, 06 Mar 2026 reply
-
RC2: 'Comment on mr-2026-3', Anonymous Referee #2, 10 Mar 2026
reply
This manuscript presents an investigation of two Gd3+ compounds to cause 19F and 13C paramagnetic relaxation enhancement of a model protein TET2. The goal is to speed up 19F and 13C T1 relaxation without excessively enhancingT2 relaxation so that one can accelerate signal-averaging of 19F and 13C direct polarization (DP) experiments while retaining spectral resolution. The experiments were rigorously conducted and the relaxation data were carefully analyzed. The differential relaxation observed for the four Trp residues in the protein is interesting, although the origin is not yet fully understood. I suggest a few changes to further improve the manuscript:
1) The authors should show 19F DP spectra of the protein bound to 8 mM Gd(DTPA-BMA) and Gd(DTPA), compared with the apo protein spectra. These should be added to the main text. Currently Fig S7 shows the severely line-broadened spectrum (T2 PRE) of the Gd(DTPA) sample but not that of the better compound, GD(DPA-BMA).
2) Likewise, the authors should show 13C spectra of the three samples: apo, 8 mM Gd(DTPA) and 8 mM Gd(DTPA-BMA). Both 13C CP and DP spectra should be shown, to illustrate the effects of the Gd3+ compounds on 13CT2 and 1H T1 relaxation.
3) Based on the 19F R1 and R2 relaxation enhancement factors measured for each Trp residue, can the authors deduce the tauc and the distance of the nearest Gd3+ dopant to each residue using equation 1? Moreover, assuming a reasonable tauc value, can the authors estimate the distance range where one can obtain significant T1 PRE but not T2 PRE to speed up experiments without suffering excessive line broadening?
Citation: https://doi.org/10.5194/mr-2026-3-RC2
Supplement
Viewed
| HTML | XML | Total | Supplement | BibTeX | EndNote | |
|---|---|---|---|---|---|---|
| 162 | 58 | 14 | 234 | 31 | 10 | 9 |
- HTML: 162
- PDF: 58
- XML: 14
- Total: 234
- Supplement: 31
- BibTeX: 10
- EndNote: 9
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1
The manuscript presents a comparison of two gadolinium complexes for enhancing the relaxation of 19F magnetization in a microcrystalline protein made with 13C-labelled fluorotryptophan. The relaxation agents were used in different concentrations to determine their optimal concentration, where longitudinal relaxation was enhanced without too much acceleration of the transverse relaxation. The work has been performed with exemplary care and all relevant details necessary for reproducing the results have been provided. It is suitable for publication with minor corrections.
To explain the differences between Gd(DTPA) and Gd (DTPA-BMA):
Could the charge of the Gd(DTPA) complex encourage binding to the protein, whereas the zero net charge of Gd(DTPA-BMA) is more likely to prevent specific binding? Inspection of the protein structure would tell the locations of positively charged amino acid residues (or overall positive electrostatic potential) in the vicinity of the tryptophan side chains.
Line 170: Are the different water exchange rates in Gd(DTPA) and Gd(DTPA-BMA) the most plausible mechanism for the different PREs, i.e., is faster water relaxation the main driving source of accelerated longitudinal 19F relaxation? In principle, the importance of water could be determined by using D2O as the solvent during the crystallization but this would add much experimental work peripheral to the scope of the present article.
Minor points:
Some of the data shown in Figures S3 and S4 seem to indicate slower relaxation in the presence of 2 or 4 mM Gd(DTPA-BMA) than in its absence (for some of the fluorotryptophan residues). Is this simply a matter of limited SNR?
In solution, the 13C relaxation of C-F groups in the tryptophan indole ring is subject to an intense TROSY effect (see, e.g., Maleckis et al., Org. Biomol. Chem., 19, 5133, https://doi.org/10.1039/D1OB00611H, 2021). Can the authors reveal anything about the 13C NMR spectra of the fluorotryptophans in the TET2 protein in solution (although this is definitely outside the scope of this article)?
Very minor points:
Line 10: it would be nice to report the name of the protein in the abstract.
Line 73: “which also comprises all plasmid details” – I presume this refers to AddGene rather than the plasmid, but is this part of the sentence necessary?
Lines 87 and 105: the names of chemicals are usually spelled with small characters.
Line 150 and elsewhere: please include the superscript ‘opt’ with tau(r.d.).
The references need to be double-checked. For example, the reference by Gronenborn appeared in Structure (line 264) and the reference by Jaroniec in Solid State Nuclear Magnetic Resonance (line 274).
Legend of Figure S8: for consistency, please provide the references in the Harvard style of referencing (not numbers) and the references themselves in the style of the main text.
Please provide the commercial source of Gd(DTPA) and Gd(DTPA-BMA). Gd(DTPA-BMA) sold under the tradename of Omniscan contains also 5% NaCa(DTPA-BMA), which is a charged complex.