Accelerated ¹⁹F biomolecular magic-angle spinning NMR with paramagnetic dopants

The advantageous characteristics attributed to the ¹⁹F nucleus have made it a popular target for nuclear magnetic resonance (NMR) once again in recent years. Aside from solution NMR, an increasing number of studies have been conducted applying solid-state magic-angle spinning (MAS) NMR to fluorine-labelled samples. Here, the high chemical shift anisotropy and strong dipolar couplings can be utilised 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-labelled biological samples. We study the effect of Gd(DTPA) and Gd(DTPA-BMA) on ¹⁹F T₁ and T₂, and ¹³C T₁ and T₂ relaxation in a [5-¹⁹F¹³C]-tryptophan-labelled 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 TET2 using a mutagenesis approach.

1 Introduction

Biomolecular ¹⁹F nuclear magnetic resonance (NMR) has regained attention in recent years due to the unique properties of the ¹⁹F nucleus and the diverse labelling strategies for proteins and nucleic acids, which make it a versatile tool for a wide range of applications and systems (Sengupta, 2024; Gronenborn, 2022; Juen et al., 2024; Overbeck et al., 2020; Heller et al., 2024). Recently, there have been exciting developments in the synthesis of compounds for the ¹⁹F-labelling of proteins (Boeszoermenyi et al., 2019; Toscano et al., 2024a, b; Suleiman et al., 2024). The introduction of a carbon-13 to form a ¹⁹F−¹³C spin pair facilitates new spectroscopic possibilities such as two-dimensional experiments and the exploration of the ¹⁹F−¹³C TROSY effect. Additional deuteration of labelling compounds can reduce the need for ¹H decoupling and lead to a reduction of unwanted relaxation pathways.

In contrast to solution-state NMR, solid-state magic-angle spinning (MAS) ¹⁹F NMR was challenging for a long time due to the high chemical shift anisotropy (CSA) of the ¹⁹F nucleus and strong dipolar ¹H−¹⁹F couplings, which can lead to severe line broadening (Ulrich, 2005). Following the development of faster spinning and specialised probe designs that enable efficient averaging and decoupling of the CSA and dipolar couplings, biomolecular ¹⁹F MAS NMR is becoming more feasible. These advancements have led to ¹⁹F NMR studies of protein microcrystals, membrane proteins, and large biomolecular assemblies such as virus capsids. The focus of these studies was set on assignments and structural investigations utilising the possibility of measuring distances of up to 20 Å (Roos et al., 2018; Duan et al., 2022; Shcherbakov et al., 2019, 2021; Porat-Dahlerbruch et al., 2022; Wang et al., 2018).

Despite the large gyromagnetic ratio of the fluorine nucleus, the sensitivity of ¹⁹F-excited MAS NMR experiments is often limited by long longitudinal T₁ relaxation times, which are often several seconds (Duan et al., 2022; Roos et al., 2018; Wang et al., 2018; Porat-Dahlerbruch et al., 2022). As the recycle delay ${\mathit{\tau }}_{\mathrm{r}.\mathrm{d}.}$ for an optimal signal-to-noise ratio (SNR) is directly related to the longitudinal relaxation of the excited nucleus (${\mathit{\tau }}_{\mathrm{r}.\mathrm{d}.}^{\mathrm{opt}}=\mathrm{1.26}\cdot T_{\mathrm{1}}$; Schanda, 2009), most of the experiment time is spent waiting for the spin polarisation to build up again. The use of deuterated precursors for ¹⁹F labelling results in even longer T₁ relaxation, as short-range ¹H−¹⁹F dipolar couplings are reduced and dipolar relaxation pathways are minimised.

Paramagnetic doping is an established and effective method to reduce the acquisition time of measurements. In solids, it has primarily been used for experiments in which protons are the initially excited nuclei. The addition of a paramagnetic compound to the sample, e.g. Cu²⁺ or Gd³⁺ chelates, results in enhanced nuclear spin relaxation. The longitudinal Γ¹ and transverse Γ² paramagnetic relaxation enhancement (PRE) is given by Solomon (1955); Bertini et al. (2001); Bloembergen and Morgan (1961); Konig (1982)

$$\begin{array}{}\text{(1)}& {\displaystyle }\begin{array}{rl}{\mathrm{\Gamma }}^{\mathrm{1}}& \approx {\displaystyle \frac{\mathrm{2}}{\mathrm{15}}}{\left({\displaystyle \frac{{\mathit{\mu }}_{\mathrm{0}}}{\mathrm{4}\mathit{\pi }}}\right)}^{\mathrm{2}}{\displaystyle \frac{{\mathit{\gamma }}_{\mathrm{I}}^{\mathrm{2}}{g}_{\mathrm{e}}^{\mathrm{2}}{\mathit{\mu }}_{\mathrm{B}}^{\mathrm{2}}S(S+\mathrm{1})}{r^{\mathrm{6}}}}\\ & \cdot \left({\displaystyle \frac{\mathrm{3}{\mathit{\tau }}_{\mathrm{c}}}{\mathrm{1}+{\mathit{\omega }}_{\mathrm{I}}^{\mathrm{2}}{\mathit{\tau }}_{\mathrm{c}}^{\mathrm{2}}}}+{\displaystyle \frac{\mathrm{7}{\mathit{\tau }}_{\mathrm{c}}}{\mathrm{1}+{\mathit{\omega }}_{\mathrm{e}}^{\mathrm{2}}{\mathit{\tau }}_{\mathrm{c}}^{\mathrm{2}}}}\right)\end{array}\text{(2)}& {\displaystyle }\begin{array}{rl}{\mathrm{\Gamma }}^{\mathrm{2}}& \approx {\displaystyle \frac{\mathrm{1}}{\mathrm{15}}}{\left({\displaystyle \frac{{\mathit{\mu }}_{\mathrm{0}}}{\mathrm{4}\mathit{\pi }}}\right)}^{\mathrm{2}}{\displaystyle \frac{{\mathit{\gamma }}_{\mathrm{I}}^{\mathrm{2}}{g}_{\mathrm{e}}^{\mathrm{2}}{\mathit{\mu }}_{\mathrm{B}}^{\mathrm{2}}S(S+\mathrm{1})}{r^{\mathrm{6}}}}\\ & \cdot \left(\mathrm{4}{\mathit{\tau }}_{\mathrm{c}}+{\displaystyle \frac{\mathrm{3}{\mathit{\tau }}_{\mathrm{c}}}{\mathrm{1}+{\mathit{\omega }}_{\mathrm{I}}^{\mathrm{2}}{\mathit{\tau }}_{\mathrm{c}}^{\mathrm{2}}}}+{\displaystyle \frac{\mathrm{13}{\mathit{\tau }}_{\mathrm{c}}}{\mathrm{1}+{\mathit{\omega }}_{\mathrm{e}}^{\mathrm{2}}{\mathit{\tau }}_{\mathrm{c}}^{\mathrm{2}}}}\right),\end{array}\end{array}$$

with the vacuum permeability μ₀, the gyromagnetic ratio of the nucleus γ_I, the electron g value g_e, the Bohr magneton μ_B, the electron spin quantum number S, the electron-nucleus distance r, the rotational correlation time τ_c, and the nuclear and electron Larmor frequencies ω_I and ω_e. The goal is to find a concentration of the paramagnetic compound in the buffer that, on the one hand, significantly accelerates ¹H T₁ relaxation, allowing a shorter recycle delay and therefore faster acquisition, and, on the other hand, does not shorten T₂, so as not to induce line broadening. While the paramagnetic effects depend on the proximity of the unpaired electron, the enhancement of longitudinal relaxation is spread across the molecule from those nuclei directly relaxed by the paramagnetic centre to other nuclei via ¹H−¹H spin diffusion (Wickramasinghe et al., 2009). The first studies to achieve a significant increase in SNR per unit time utilised Cu²⁺-EDTA as a dopant, which remains widely used to date (Wickramasinghe et al., 2007, 2009). However, the higher PRE effect of Gd³⁺ chelates, such as Gd(DOTA), Gd(DTPA-BMA), or Gd(DTPA), enables the use of lower concentrations of the compound, reducing possible interactions with the studied biomolecule and sample heating (Linser et al., 2007; Ullrich et al., 2014; Mroue et al., 2014; Öster et al., 2019).

Even though paramagnetic doping was mainly applied for its effect on the T₁ relaxation of protons, it also increases the relaxation of other nuclei, with the strength of the effect being proportional to the squared gyromagnetic ratio of the nucleus (Eq. 1). Due to the high gyromagnetic ratio of ¹⁹F, a significant reduction in measurement time can be expected for biomolecular ¹⁹F MAS NMR experiments with similar concentrations of paramagnetic compounds, as are used for proton-excited experiments.

Transversal and longitudinal PREs of fluorine in solution were measured previously to obtain distance restraints between a fluorine atom and a paramagnetic moiety (Shi et al., 2012; Matei and Gronenborn, 2016; Bondarenko et al., 2019; Huang et al., 2020). In solids, Lu et al. (2020) demonstrated paramagnetic doping with Cu²⁺ ions in the context of fluorinated crystalline pharmaceuticals for structural characterisation by ¹⁹F MAS NMR. They achieved a significant reduction of the ¹⁹F T₁ relaxation times in their samples, resulting in approximately 2.5 times faster acquisition of fluorine NMR spectra, which highlights the potential benefits of combining paramagnetic doping and ¹⁹F solid-state MAS NMR (Lu et al., 2020).

In this work, we explore the potential benefits of paramagnetic doping for biomolecular ¹⁹F MAS NMR on proteins, which has not been reported so far, to the best of our knowledge. Using the deuterated 5-fluorotryptophan-labelled protein TET2 (12×39 kDa), we measure ¹⁹F T₁ and T₂, and ¹³C T₁ and T₂ as a function of the concentration of two Gd³⁺ complexes, Gd(DTPA-BMA) and Gd(DTPA). We find that a concentration of 8 mM Gd(DTPA-BMA) is optimal to reduce measurement times of ¹⁹F MAS NMR experiments through a decrease in the ¹⁹F T₁ relaxation time without significant line broadening.

2 Methods

2.1 Protein production and purification

The aminopeptidase TET2 from Pyrococcus horikoshii (UniProt entry O59196) was produced via overexpression of the pET41c-PhTET2 plasmid in Escherichia coli BL21(DE3) RIL cells. The plasmid is available from AddGene (https://www.addgene.org/182428/, last access: 15 February 2026). For the resonance assignment, tryptophans were mutated into phenylalanines in different combinations either as single mutant (mutant 1: W106F) or as triple mutants (mutant 2 (only W106): W136F, W164F, W276F; mutant 3 (only W136): W106F, W164F, W276F; mutant 4 (only W164): W106F, W136F, W276F; mutant 5 (only W276): W106F, W136F, W164F). The ¹⁹F labelling was achieved either with 5-fluoroindole (Sigma-Aldrich, catalogue number F9108) in protonated medium (mutants) or with [$\mathrm{5}{-}^{\mathrm{13}}\mathrm{C},\mathrm{3},\mathrm{4},\mathrm{6}{-}^{\mathrm{2}}{\mathrm{H}}_{\mathrm{3}}$]-5-fluoroanthranilic acid (5FC-anthranilic acid) in deuterated medium (wild type).

The 5-fluoroanthranilic acid isotopologue was synthesised in-house by adapting the synthetic route reported by Suleiman et al. (2024) to the present labelling scheme; details of the synthesis will be published elsewhere.

The plasmid (kanamycin resistance) was transformed into competent BL21(DE3) RIL cells (chloramphenicol resistance) via heat shock. Unless otherwise mentioned, all cultures contained kanamycin and chloramphenicol, and shaking was performed at 200 rpm and 37 °C.

5-fluoroindole labelling was achieved as follows. After precultures in LB medium and minimal M9 medium, the main culture was inoculated to an optical density at 600 nm (OD₆₀₀) of 0.2 and shaken until it reached 0.6–0.7. At this point, 1 g L⁻¹ glyphosate (abcr, Karlsruhe, Germany; catalogue number AB505195) was added, and the culture was grown for 15 min before 100 mg L⁻¹ 5-fluoroindole, 50 mg L⁻¹ L-tyrosine (Sigma T3754), and 50 mg L⁻¹ L-phenylalanine (Sigma 78019) were added. The culture was grown for 45 min, and expression was induced with 1 mM isopropyl-β-D-thiogalactopyranosid (IPTG). Cells were harvested at 6500 rcf for 15 min after 4 h of shaking.

Labelling with 5FC-anthranilic acid was achieved as follows. Cells were adjusted to deuterated M9 medium by growth in consecutive precultures of LB medium and M9 medium prepared with 100 % H₂O, 50 % H₂O/50 % D₂O, and 100 % D₂O. The final preculture and the main culture were prepared with ¹⁵NH₄Cl and D-²H₇-glucose. The main culture was inoculated to an OD₆₀₀ of 0.2 and shaken until it reached 0.6–0.7. At this point, 1 g L⁻¹ glyphosate, 50 mg L⁻¹ L-tyrosine, 50 mg L⁻¹ L-phenylalanine, and 15 mg L⁻¹ 5FC-anthranilic acid were added, and the culture was shaken for 40 min. The temperature was then reduced to 28 °C for 15 min before induction with 1 mM IPTG. Cells were grown overnight at 28 °C and then harvested at 6500 rcf for 15 min.

The cell pellet was re-suspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05 mg mL⁻¹ DNase, 2 mM MgCl₂, 0.025 mg mL⁻¹ RNase, and 0.5 tablets cOmplete EDTA-free protease inhibitor), kept on ice for 30 min, and sonicated for 2 min. A heat shock was performed at 80 °C for 15 min. After the addition of 10 mL buffer A (20 mM Tris-HCl pH 7.5, 100 mM NaCl), the cell debris was collected by centrifugation at 46 000 rcf for 40 min at 4 °C. The supernatant was washed with buffer A using an Amicon ultra centrifugal filter with a molecular weight cutoff of 100 kDa before loading onto a RESOURCE Q column (Cytiva) and eluted with a gradient over 10 column volumes from buffer A to buffer B (20 mM Tris-HCl pH 7.5, 1 M NaCl). The fractions containing the protein were concentrated, loaded onto a HiLoad 10/300 Superdex 200 pg column (Cytiva), and eluted in buffer A.

2.2 Sample preparation

Samples for solid-state MAS NMR were prepared by batch crystallisation of TET2 with 2-methyl-2,4-pentanediol (MPD; Sigma 68340) (Gauto et al., 2019a). A solution of 10 mg mL⁻¹ protein and the paramagnetic compound (where applicable; Gd(DTPA): Sigma 381667, Gd(DTPA-BMA): GE Healthcare Omniscan (contains 4.2 % NaCa(DTPA-BMA)) in buffer A was mixed with MPD in a $v/v$ ratio of 1:1. The concentration given for the paramagnetic compound in the following is related to the final concentration in the sample, including MPD. The microcrystals were filled into a 1.3 mM MAS rotor (Bruker) by ultracentrifugation overnight at 4 °C and 68 000 rcf.

2.3 NMR

MAS NMR experiments were performed on a Bruker Avance Neo spectrometer operating at 14.09 T (600 MHz ¹H Larmor frequency). A triple-resonance HFX probe head from PhoenixNMR equipped with a 1.3 mm MAS stator from Bruker was used, with the X channel tuned to ¹³C. Temperature calibration was done with an external ²H₄-methanol sample (Karschin et al., 2022), and chemical shift referencing was done indirectly via the ¹H signal of 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS). All experiments were performed at a MAS frequency of 55.555 kHz and a sample temperature of approximately 309 K. Spectra were processed with Bruker Topspin software (versions 4.1.4 and 4.5.0).

Pulse sequence diagrams can be found in Fig. 1. All experiments were performed with ¹⁹F detection (10 ms acquisition time), and ¹H and ¹³C decoupling unless stated otherwise. Composite pulse decoupling during acquisition was typically achieved with 10 kHz swfTPPM (Thakur et al., 2006) on ¹H and 10 kHz WALTZ-16 (Shaka et al., 1983) on ¹³C. The recycle delay was set to $\approx \mathrm{1.3}\cdot T_{\mathrm{1}}$ of ¹⁹F, depending on the concentration of the paramagnetic compound, except for the ¹⁹F saturation recovery experiment, in which the recycle delay was set to 1.2 s. The pre-saturation block in the saturation recovery experiment was repeated n=50 times with a delay Δ=4.5 ms. Magnetisation transfer in ¹³C relaxation experiments was achieved via dipolar ¹⁹F−¹³C cross-polarisation (CP) steps. Typical CP spin-lock radio-frequency field strengths were 40 kHz on ¹³C and 90 kHz on ¹⁹F, with a linear ramp of 90 %–100 % and a transfer time of 400 µs. ¹⁹F and ¹³C relaxation experiments were recorded as pseudo two-dimensional spectra with one ¹⁹F frequency dimension and one pseudo dimension in which a relaxation delay τ was incremented.

Figure 1 Pulse sequences used in this study. Closed, open, and wide-open rectangles denote 90°, 180°, and CP spin-lock pulses, respectively. Grey rectangles indicate composite pulse decoupling. Δ and τ are delays, and n indicates a loop. Pulse phases are indicated above the pulse, with Φ_n marking a pulse undergoing phase cycling, as noted below. Acquisition is denoted with a free induction decay scheme with the receiver phase Φ_rec,n indicated above. p-2D stands for pseudo two-dimensional spectrum with one frequency dimension and one pseudo dimension in which a delay τ is incremented.

2.4 Relaxation rate analysis

Spectra were processed as pseudo two-dimensional spectra in Topspin and converted to UCSF format with the bruk2ucsf program provided in Sparky (Goddard and Kneller, 2008). Python scripts for analysis of relaxation rate constants were written in-house, utilising the Nmrglue package (Helmus and Jaroniec, 2013). Spectra were split into individual 1D slices, and peaks were fitted with the routine implemented in Nmrglue. The intensities were then fitted to a mono-exponential function. Errors were determined by Monte Carlo analysis (500 iterations) using one standard deviation of the spectral noise.

3 Results and discussion

3.1 Assignment of fluorine-labelled tryptophans in TET2

TET2 is a dodecameric aminopeptidase from P. horikoshii that has been studied extensively by MAS NMR previously (Gauto et al., 2019b, 2022). Each of the 12 identical subunits contains four tryptophan residues: W106, W136, W164, and W276 (Fig. 2a). To achieve fluorine labelling of the aromatic ring, we expressed TET2 in deuterated M9 medium and added 5-fluoroanthranilic acid, which is metabolised by the bacteria into 5-fluoro-L-tryptophan (Fig. 2b). This precursor has a ¹⁹F−¹³C spin pair at position 5 in the aromatic ring and is deuterated at positions 3, 4, and 6. We will refer to the labelled protein as 5FC-W-TET in the remainder of this discussion. As expected, the ¹⁹F MAS NMR spectrum of 5FC-W-TET shows four individual peaks (Fig. 2c, upper spectrum). For the assignment of the signals, we prepared five Trp to Phe mutants in non-deuterated medium using the commercially available precursor 5-fluoroindole (see the Methods section for details). The respective spectra show either one (triple mutants) or three (single mutant) signals, which allowed us to assign the four signals (Fig. 2c, bottom spectra). Note that the spectrum of the deuterated wild type is shifted due to an isotope shift (Luck et al., 1996).

Figure 2 (a) Structure of the dodecameric TET2 (PDB: 1Y0R) (Borissenko and Groll, 2005) in cartoon representation with one subunit highlighted in beige (upper panel). The lower panel shows a close-up of one subunit with the four tryptophans indicated as orange sticks. Position 5 in the tryptophan ring is highlighted with spheres. (b) Structure of 5-fluoroanthranilic acid (left), which is converted into 5-fluoro-L-tryptophan (right) by the bacteria. (c) 1D ¹⁹F MAS NMR spectrum of 5FC-W-TET. The upper panel displays the spectrum of the wild type, showing all four tryptophan peaks. The lower panel shows spectra of the five assignment mutants (mutant 1: W106F; mutant 2: W136F, W164F, W276F; mutant 3: W106F, W164F, W276F; mutant 4: W106F, W136F, W276F; mutant 5: W106F, W136F, W164F; see the Methods section for details). The resulting assignment is indicated at the top. The wild-type spectrum is shifted due to the isotope shift, as this sample is deuterated compared to the mutants. (d) Exponential fits of ¹⁹F saturation recovery experiments on 5FC-W-TET without paramagnetic dopant. (e) Structure of Gd(DTPA-BMA). (f) Structure of Gd(DTPA).

3.2 The effect of Gd(DTPA-BMA) and Gd(DTPA) on bulk ¹⁹F and ¹³C relaxation

In recent years, several studies have been published using biomolecular ¹⁹F MAS NMR to study structural aspects of proteins (Roos et al., 2018; Duan et al., 2022; Shcherbakov et al., 2019, 2021; Porat-Dahlerbruch et al., 2022; Wang et al., 2018). The ¹⁹F T₁ relaxation time was often reported to be several seconds long, leading to a long ${\mathit{\tau }}_{\mathrm{r}.\mathrm{d}.}^{\mathrm{opt}}$ to obtain an optimal SNR. We measured ¹⁹F T₁ of 5FC-W-TET with a saturation recovery experiment (Fig. 2d), and obtained values between 1.67±0.08 s (W164) and 6.0±0.4 s (W136). Considering the highest value, this would correspond to a recycle delay of ${\mathit{\tau }}_{\mathrm{r}.\mathrm{d}.}^{\mathrm{opt}}=\mathrm{7.56}\mathrm{s}$.

To reduce the ¹⁹F T₁, we used paramagnetic doping with two different Gd³⁺ chelates. We prepared samples with six concentrations of Gd(DTPA-BMA) (0, 2, 4, 6, 8, and 16 mM, Fig. 2e), and measured ¹⁹F T₁ and T₂, and ¹³C T₁ and T₂ for each residue (Figs. S1, S2, S3, S4, S6 in the Supplement). For Gd(DTPA) (Fig. 2f), we prepared samples with 2 and 8 mM of the compound and measured ¹⁹F T₁ and T₂ (Fig. S5, S6). The bulk relaxation rates $R=T^{-\mathrm{1}}$ (average over all residues) are shown in Fig. 3a–d.

At 8 mM Gd(DTPA), the increase in R₂ led to a broadening of the whole spectrum that made the individual peaks indistinguishable (Fig. S7). Additionally, Gd(DTPA) changed the crystallisation behaviour of TET2, possibly due to binding to the protein surface (Fig. S8) (Petros et al., 1990). We therefore refrained from preparing samples with other concentrations or measuring ¹³C relaxation rate constants.

We find that both compounds lead to an increase in R₁ as well as R₂. To quantify the effect of the two compounds, we determined the longitudinal and transverse PREs (Γ₁ and Γ₂), which are given by the slope of a linear fit of the respective relaxation rate constants as a function of the concentration of paramagnetic dopant. We performed fits of the bulk and per-residue relaxation rate constants to obtain the PREs for Gd(DTPA-BMA) (¹⁹F and ¹³C; Fig. 3e–f) and for Gd(DTPA) (only ¹⁹F; Fig. 3g–h).

Figure 3 ¹⁹F and ¹³C longitudinal and transverse PRE (Γ₁ and Γ₂) for Gd(DTPA-BMA) and Gd(DTPA) measured on 5FC-W-Trp. (a–b) Linear fits of bulk ¹⁹F R₁ (a) and R₂ (b) relaxation rate constants as a function of the concentration of Gd(DTPA-BMA) (blue) and Gd(DTPA) (yellow). (c–d) Linear fits of bulk ¹³C R₁ (c) and R₂ (d) relaxation rate constants as a function of the concentration of Gd(DTPA-BMA) (light blue). (e–f) ¹⁹F (dark blue) and ¹³C (light blue) Γ₁ (e) and Γ₂ (f) for Gd(DTPA-BMA). (g–h) ¹⁹F Γ₁ (g) and Γ₂ (h) for Gd(DTPA). The bulk relaxation rate constants in (a)–(d) are the average over the four residues (see Figs. S1, S2, S3, S4, S5) except for the rate at 8 mM Gd(DTPA), which was only measured as a bulk rate due to line broadening. In (e)–(h), the first bar of each plot is the bulk PRE resulting from the fits in (a)–(d) (grey background), followed by the individual values for each residue resulting from the fits in Figs. S1, S2, S3, S4, S5. Note that the per-residue fits for Gd(DTPA) (g, h) are performed with only two points (0 and 2 mM), while the bulk fit is performed with three points (0, 2, and 8 mM). This leads to a deviation between Γ^bulk and the average over the residue-wise values.

As expected, both Γ₁ and Γ₂ are smaller for ¹³C than for ¹⁹F. This is due to the smaller gyromagnetic ratio of the carbon nucleus (Eq. 1).

Interestingly, the measured ¹⁹F PREs for Gd(DTPA) are much higher than for Gd(DTPA-BMA). The bulk effect of Gd(DTPA) on R₁ (${\mathrm{\Gamma }}_{\mathrm{1}}=\mathrm{0.743}\pm \mathrm{0.015}{\mathrm{s}}^{-\mathrm{1}}{\mathrm{mM}}^{-\mathrm{1}}$) is roughly seven times stronger than the effect of Gd(DTPA-BMA) (${\mathrm{\Gamma }}_{\mathrm{1}}=\mathrm{0.101}\pm \mathrm{0.028}{\mathrm{s}}^{-\mathrm{1}}{\mathrm{mM}}^{-\mathrm{1}}$). For Γ₂, the difference is even bigger: The bulk value for Gd(DTPA) (${\mathrm{\Gamma }}_{\mathrm{2}}=\mathrm{556.2}\pm \mathrm{0.7}{\mathrm{s}}^{-\mathrm{1}}{\mathrm{mM}}^{-\mathrm{1}}$) is over 14 times higher than the value for Gd(DTPA-BMA) (${\mathrm{\Gamma }}_{\mathrm{2}}=\mathrm{38.2}\pm \mathrm{0.6}{\mathrm{s}}^{-\mathrm{1}}{\mathrm{mM}}^{-\mathrm{1}}$). The differences in relaxation behaviour are likely the result of the specific physicochemical properties of the two compounds (e.g. the slower water exchange rate of Gd(DTPA-BMA)) (Caravan et al., 1999).

We find that in our case, paramagnetic doping with 8 mM Gd(DTPA-BMA) is the best compromise between a significant reduction of ¹⁹F T₁ without heavily compromising T₂ and therefore spectral resolution. The decrease in bulk T₁ from 3.2±1.3 s at 0 mM to 0.92±0.25 s at 8 mM Gd(DTPA-BMA) translates to a reduction of ${\mathit{\tau }}_{\mathrm{r}.\mathrm{d}.}^{\mathrm{opt}}$ from 4.0±1.6 to 1.2±0.4 s. The more than 3-fold shorter recycle delay significantly reduces the measurement times of ¹⁹F MAS NMR spectra, or, in other words, increases the SNR per unit time. Although the same effect could be achieved with lower concentrations of Gd(DTPA), we prefer the use of Gd(DTPA-BMA) due to a potential interaction of Gd(DTPA) with the protein in our case (see above). Figure 4 shows a comparison of the ¹⁹F spectra of 5FC-W-TET with and without dopant (¹³C spectra are shown in Fig. S9).

Figure 4 ¹⁹F MAS NMR spectrum of 5FC-W-TET without dopant (grey), with 8 mM Gd(DTPA-BMA) (blue), and with 2 mM Gd(DTPA) (yellow). ${\mathit{\tau }}_{\mathrm{r}.\mathrm{d}.}$ was set to five times the ¹⁹F T₁ of the slowest relaxing peak for each sample. Note that the absolute intensities between samples are not comparable, as the amount of protein inside the rotor is hard to determine.

3.3 The relative PREs of individual tryptophan residues

The observed PREs for the individual tryptophans differ (Fig. 3e–h), which can be expected as they are impacted by a multitude of factors, such as the solvent accessibility of the residue, its dynamics, or the density of surrounding protons (Tang et al., 2011; Wickramasinghe et al., 2009). The influences of these factors can conversely even be used to characterise the surfaces and interaction interfaces of proteins with so-called solvent PREs (Pintacuda and Otting, 2002; Öster et al., 2017; Hocking et al., 2013).

To rationalise the observed PRE data for the four Trp sites, we compared them to structural parameters. We reasoned that the relaxation properties may be impacted by the ¹H spins surrounding each of the ¹⁹F Trp sites and calculated the root-sum-square dipolar coupling d^rss (Fig. S10a). This parameter approximates the effective dipolar coupling network and can serve as an indicator for spin diffusion, which influences the propagation of the PRE effect throughout the protein. It was calculated as the square root of the sum of squared dipolar couplings between a fluorine and all back-exchangeable protons (Zorin et al., 2006). Moreover, we calculated the solvent accessible surface area (SASA) of each tryptophan (Fig. S10b), which is an approximate measure of the shortest distance between the paramagnetic compound and a given ¹⁹F atom. In our case, we did not find a direct correlation of these parameters with the observed PREs (Fig. S10c–d).

Interestingly, the PRE patterns (relative strength of the measured PREs for the four residues) are different for different relaxation rate constants (Γ₁ and Γ₂), different compounds, and different nuclei (¹⁹F and ¹³C) (Fig. 3e–h). In addition to the factors mentioned above, other influencing parameters that could explain these different patterns include a lower spin diffusion efficiency for ¹³C, specific binding of the Gd³⁺ complex to the protein, and properties of the compound, such as the rotational correlation time τ_c or the water exchange rate.

Differences between the patterns observed for Gd(DTPA-BMA) and Gd(DTPA) could be due to the specific binding of one of the complexes to the protein. A decrease in the distance r between the compound and residues close to the binding site would increase the observed PRE effects as both Γ₁ and Γ₂ are proportional to r⁻⁶ (Eq. 1). The binding would also decrease the τ_c of the compound, which could, in combination with the dynamics of a specific tryptophan, lead to dampening or acceleration of the PRE.

The most striking observation is that the ¹⁹F Γ¹ of W164 is very high relative to the other residues compared to ¹⁹F Γ² for both Gd³⁺ complexes. Such a difference between the longitudinal and transverse PRE can also be a result of a local reduction of τ_c due to binding. As Γ₁ and Γ₂ sample the spectral density at different frequencies, their reaction to changes of τ_c is not the same (Eq. 1) (Jaroniec, 2012). The complex interplay of diverse parameters makes it difficult to understand these patterns in detail.

4 Conclusions

The potential of biomolecular ¹⁹F MAS NMR is often limited by long T₁ relaxation times that require recycle delays of several seconds. In this work, we discussed the application of paramagnetic doping with Gd³⁺ complexes to accelerate these experiments. Previously, paramagnetic doping with different compounds was used to reduce the ¹H T₁ in a variety of sample types, such as membrane proteins and protein microcrystals (Wickramasinghe et al., 2007, 2009; Linser et al., 2007; Ullrich et al., 2014). To our knowledge, this is the first study applying paramagnetic doping for MAS NMR to fluorine-labelled biological samples.

We evaluated the effect of two different Gd³⁺ complexes – Gd(DTPA-BMA) and Gd(DTPA) – on the ¹⁹F T₁ and T₂, and ¹³C T₁ and T₂ relaxation times in deuterated and 5-fluorotryptophan-labelled TET2. The addition of 8 mM Gd(DTPA-BMA) reduces ${\mathit{\tau }}_{\mathrm{r}.\mathrm{d}.}^{\mathrm{opt}}$ by a factor of more than 3 compared to the undoped sample, without causing significant line broadening. The addition of Gd(DTPA) results in a stronger paramagnetic relaxation enhancement, but it is a less favourable compound due to its interaction with the protein.

We anticipate that the use of paramagnetic doping for biomolecular ¹⁹F MAS NMR can be applied to a variety of systems, experiments, and different types of sample preparations. The increase in sensitivity will be especially beneficial for structural studies and the measurement of anisotropic spin interactions. As for paramagnetic doping of non-fluorine-labelled samples, the optimal concentration and compound are likely to depend on the specific experimental setup.