# Fast ultra-selective 1H-15N 1D NMR spectroscopy unlocks atom-resolved dynamics of low-complexity protein regions

Wiktor Adamski, Géraldine Levy, François-Xavier Cantrelle, Davy Sinnaeve

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Wiktor Adamski, Géraldine Levy, François-Xavier Cantrelle, Davy Sinnaeve. Fast ultra-selective 1H-15N 1D NMR spectroscopy unlocks atom-resolved dynamics of low-complexity protein regions. 2025. [hal-05393364](https:// HAL Id: hal-05393364 )

Preprint submitted on 2 Dec 2025

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# Fast ultra-selective ¹H-¹⁵N 1D NMR spectroscopy unlocks atom-resolved dynamics of low-complexity protein regions

Wiktor Adamski<fnref n="1" />,<fnref n="2" />*, Geraldine R. Levy<fnref n="1" />,<fnref n="2" />, François-Xavier Cantrelle<fnref n="1" />,<fnref n="2" /> and Davy Sinnaeve<fnref n="1" />,<fnref n="2" />*

* Corresponding authors:
wiktor.adamski1992@gmail.com
davy.sinnaeve@univ-lille.fr

## ABSTRACT

Insight into the conformational dynamics of proteins is essential towards understanding their function at a molecular level. The motions experienced by individual atoms in the protein can be precisely quantified through NMR relaxation rates, but their measurement requires well-resolved spectral responses. Two-dimensional ¹H-¹⁵N correlation spectra are the standard approach to resolve amide signals in protein NMR, but come with an excessive cost in experimental time when spectra are heavily congested due to limited ¹⁵N chemical shift dispersions. This limitation often thwarts complete characterization of dynamics for intrinsically disordered proteins, especially when they feature low-complexity or homopolymer amino acid sequences. Here, we introduce an ultra-selective ¹H-¹⁵N NMR method that allows high-quality measurement of individual ¹⁵N spin-relaxation constants using fast 1D NMR spectra, even when ¹⁵N resonances are merely 6-8 Hz apart. We demonstrate the new experiment by characterizing, for the first time, pico- to nanosecond dynamics along a 16-residue polyglutamine stretch within the protein huntingtin, the causal agent of Huntington's disease, as well as millisecond conformational exchange in the SH3GL3 protein. The new experiment will find wide application in the study of conformational dynamics of intrinsically disordered proteins or any other biomacromolecule that features highly dense ¹H-¹⁵N 2D spectra.

<footnote n="1">¹ CNRS EMR 9002 – Integrative Structural Biology, F-59000, Lille, France.</footnote>

<footnote n="2">² Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, UMR 1167 – RID-AGE – Risk Factors and Molecular Determinants of Aging-Related Diseases, F-59000, Lille, France.</footnote>
Conformational dynamics are key for protein function. To quantify the conformational motions sensed by individual nuclei, NMR relaxation rates are a primary information source.¹ For this, it is essential that NMR spectral responses are fully resolved, which is usually achieved with multidimensional heteronuclear experiments. Unfortunately, multidimensional acquisition comes at the price of increased minimal experimental time. The smaller the chemical shift differences in the indirect dimension, the more time-consuming the experiment becomes. The time-cost is exacerbated for relaxation measurements, as these necessitate recording 2D experiments about ten times with variable delays, radio-frequency (rf) amplitudes or magnetic field strength.¹a,¹b When spectra are congested and chemical shift differences are very small — rampant for intrinsically disordered proteins (IDPs) with low sequence-complexity — complete high-quality quantification of conformational dynamics would require an excessive experimental time.

The exon-1 fragment of the protein huntingtin (htt) presents a clear illustration of this issue. It features a 16-residue polyglutamine (polyQ) homorepeat, the abnormal expansion of which triggers Huntington's disease.² A gradual change of secondary structure adopted along the polyQ has been proposed as central towards understanding pathogenicity.³ Mapping the conformational dynamics along this tract by NMR relaxation analysis would be a logical pursuit, but until now the profound density of its 2D ¹H-¹⁵N HSQC (Figure 1a) has obstructed relaxation measurements on individual glutamines.³b Note that the 2D HSQC in Figure 1a was recorded with an unusually high digital resolution in the ¹⁵N dimension, though much overlap remains. Although relaxation rates have been reported for other polyglutamine-containing proteins within reasonable experimental times using 2D NMR and minimal signal averaging,⁴ reliable and precise quantification of conformational dynamics requires high-quality spectral data with sufficient signal-to-noise ratios along the full relaxation decay curve. Given the limited concentrations imposed by the aggregation-prone htt-exon1 protein, attaining both adequate signal-to-noise and high spectral resolution is not feasible in this way (see supporting information). An existing method for resolving individual residues in polyQ sequences is through additional ¹³C isotope labelling and non-uniformly sampled (NUS) high-dimensional experiments,³b,⁴a but spectra reconstructed from NUS data result in significantly lower fidelity for relaxation analysis.⁵ Another strategy involves residue-specific ¹⁵N isotopic labeling,³a,⁶ but these methods have insufficient protein yields for relaxation measurements and it would require a separate protein production per residue. An ideal approach would isolate individual correlations from crowded signal densities without resorting to NUS methods or elaborate isotope labeling and sample productions, but with short minimal experimental times and sufficient sensitivity for relaxation measurements.

Here, we introduce a new NMR approach that meets these requirements, coined SNIPER (Selected Nuclei Irradiation for Precise and Expedited Relaxation measurements). It cleanly delivers individual ¹H-¹⁵N correlations even when they are separated by very small ¹⁵N frequency differences. Several examples yielding individual glutamine correlations from htt exon-1 are shown overlayed on the standard ¹H-¹⁵N HSQC in Figure 1a, including from within the polyQ signal density and some that were not cleanly resolved in the standard 2D HSQC (Q18/Q20 and Q48/Q59). The spectral purity and sensitivity are highly satisfactory (Figure 1b), clearing the way to measure relaxation via fast 1D acquisition (Figure 1c). Using 1D SNIPER, acquisition of a full ¹⁵N R₁, R₂ and {¹H}-¹⁵N nOe dataset took between 7.5 and 33 hours depending on the chosen residue, a fraction of the time that would be needed to acquire a standard 2D spectrum that reaches adequate ¹⁵N digital resolution and similar signal-to-noise ratios (see supporting information). Although the principle of ¹⁵N selective 1D experiments was already proposed for folded proteins with well-dispersed spectra with the aim of
accelerating off-resonance R₁p relaxation dispersion experiments (wherein the spin-lock's weak rf-field limits the validity of the experiment to a narrow ¹⁵N region, making 2D broadband acquisition particularly time-inefficient),⁷ such outstanding ¹⁵N selectivity has not yet been achieved. This parallels the recent breakthrough of ultra-selective 1D methods in small molecule ¹H NMR as a fast alternative for 2D methods.⁸

<figure><img src="image_2.png" /><figcaption>**Figure 1.** (a) Amino acid sequence of htt exon-1 polyQ-16 and a standard ¹H- ¹⁵N 2D HSQC (900 MHz, 340 µM, pH 6.8, 283 K) (grey), overlayed with several 2D SNIPERs targeting individual glutamine correlations from the polyQ (blue) or the proline-rich region (red). (b) F₁-cross-sections taken from the 2D SNIPER spectra (full line) and the standard 2D HSQC (dashed line) for a number of glutamines. (c) 1D SNIPER spectra (full line) and F₂-cross-sections from the standard 2D HSQC (dashed line) for a number of glutamines. The supporting information contains more examples.</figcaption></figure>

Figure 2 shows ¹⁵N relaxation parameters extracted from a broad selection of glutamine signals using SNIPER. The fast 1D acquisition permitted sufficient signal averaging to attain good signal-to-noise ratios throughout the entire relaxation decay curves, resulting in high-quality mono-exponential fittings (Figures S15-S16). Within the polyQ, the R₂ rates appear to be steady and high between Q18 and Q22, substantiating previous work that revealed Q20 and Q21 participate in bifurcated hydrogen bonding interactions with S16 and F17, respectively, stabilizing an α-helical secondary structure.⁶b,⁹ The subsequent monotonic descent of R₂ rates towards the C-terminal end of the polyQ implies a gradual increase in the relative importance of motions occurring on single nanosecond time-scales.¹⁰ This agrees with previous chemical shift analysis studies indicating that the α-helical propensity gradually decreases along the polyQ.³,⁶ The R₁ rates fluctuate along the polyQ, suggesting a diversity of motions on single nanosecond timescales. The {¹H}-¹⁵N nOes drop markedly beyond Q26, pointing towards a relative increase in the importance of motions occurring on the order of hundreds of picosecond time-scale.¹⁰ This indicates a loss of cooperative motions in the C-terminal segment of the polyQ and thus lack of stable secondary structures. This is consistent with previous conclusions based on chemical shifts that suggested the helical structure is almost entirely vanished at Q28 for the htt polyQ-16 sequence.⁶b For the glutamines in the
proline-rich region (Q45-Q61), a remarkably non-uniform picture emerges for all relaxation parameters. This suggests a diversity of motional behaviors that could be due to yet unidentified specific interresidue contacts and local secondary structures.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** $^{15}$N R$_{1}$, R$_{2}$ rates and { $^{1}$H}-$^{15}$N nOes (with 95% uncertainties) extracted from htt exon-1 glutamines using 1D SNIPER.</figcaption></figure>

The SNIPER approach is equally useful for fast quantification of millisecond conformational exchange. In
standard 2D zz-exchange experiments, ground and excited states feature as separate ‘diagonal’ peaks with
‘cross-peaks’ between them,¹¹ as illustrated for the SH3 domain of the SH3GL3 protein¹² in Figure 3a. This
quadrupling of the number of correlations often delivers congested zz-exchange spectra, even for folded
proteins. When the $^{15}$N chemical shifts of ground and excited states are close, long experimental times would
be needed to separate diagonal and cross-peaks in a series of standard 2D spectra with variable exchange
delays.¹¹ Using SNIPER, the same information is cleanly obtained using just two series of 1D spectra
(Figure 3b), drastically speeding up quantification of the exchange rates.
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 3.** (a) Part of a ¹H-¹⁵N 2D zz-exchange spectrum¹¹ (750 ms mixing time) of the SH3 domain of SH3GL3, showing ground and excited state correlations of residue C5 with very close ¹⁵N chemical shifts. (b) 1D SNIPER zz-exchange spectra measured with 0 and 300 ms mixing times, selecting the ¹⁵N chemical shifts of the C5 ground and excited states. (c) 1D SNIPER zz-exchange peak intensities as a function of mixing time (dots). The fitted curves assume two-site exchange (see supporting information).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 4.** SNIPER pulse sequences for measuring <sup>15</sup>N R<sub>1</sub> and R<sub>2</sub> (via constant-time R<sub>1p</sub>)<sup>7</sup> rates or zz-exchange. Thin and wide filled black rectangles are hard 90° and 180° pulses, respectively. The <sup>15</sup>N selective 90° shaped pulse is colored green. White sine-bells are <sup>1</sup>H selective 90° pulses set to the water signal. Dark blue boxes are <sup>1</sup>H and <sup>15</sup>N irradiations at matched rf-field strength (ω<sub>1</sub> = 0.5×J<sub>NH</sub>) for selective polarization transfer. Grey rectangles represent CW irradiation of the amide proton. The delay ζ is 0-5 ms, Δ is 5-40 ms, δ<sub>a</sub> is (4J<sub>NH</sub>)<sup>-1</sup> (2.65 ms), δ<sub>b</sub> is 2.35 ms. χ is a delay matched to the <sup>1</sup>H CW power so the amide proton and water magnetizations are simultaneously aligned with the CW effective B<sub>1</sub>-field.<sup>13</sup> The <sup>13</sup>C<sup>a</sup> and <sup>13</sup>C<sup>†</sup> pulses are 180° pulses. <sup>1</sup>H<sup>N</sup> selective inversion pulses are shown as grey shapes. The <sup>15</sup>N R<sub>1p</sub> spin-lock (SL) is applied on-resonance, temperature compensation spin-locks (TC) far off-resonance. The <sup>15</sup>N open-bar rectangles are pulses with flip angles matched to the spin-lock's offset from the <sup>15</sup>N resonance of interest.<sup>7</sup> Grey trapezoids are pulsed field gradients. RRI means refocused reverse INEPT. CPD denotes composite pulse decoupling. The supporting information contains phase cycling, gradient amplitudes and further technical details.</figcaption></figure>

Figure 4 shows the SNIPER pulse sequences for <sup>15</sup>N R<sub>1</sub>, R<sub>2</sub> and zz-exchange measurements (the {<sup>1</sup>H}-<sup>15</sup>N nOe sequence is found in Figure S12). It applies selective in-phase <sup>1</sup>H-<sup>15</sup>N polarization transfers using matched <sup>1</sup>H and <sup>15</sup>N irradiation, either applied simultaneously (Hartmann-Hahn, HaHa) or consecutively (refocused single field polarization transfer, rSFPT).<sup>7,14</sup> This already provides a selectivity of ca. 35-45 Hz in both <sup>1</sup>H and <sup>15</sup>N (see supporting information), but this is insufficient for very dense spectra. We therefore implemented a <sup>15</sup>N chemical-shift-selective filter (CSSF), consisting out of a 90°<sub>y</sub> - ζ - 90°<sub>-y</sub> sequence applied on longitudinal <sup>15</sup>N magnetization during <sup>1</sup>H continuous wave (CW) decoupling, followed by a purge gradient. Previous selective methods used the ζ-delay to remove <sup>15</sup>N signals found 1/4ζ Hz off-resonance,<sup>7</sup> but this results in unacceptably high relaxation losses for very small frequency differences. SNIPER rather achieves its ultra-high <sup>15</sup>N selectivity by replacing the first <sup>15</sup>N 90° pulse in the CSSF by a selective excitation pulse. To cleanly differentiate close <sup>15</sup>N frequencies, this pulse should feature a sharp excitation profile, without off-resonance excitation side-bands, while being as efficient as possible in terms of relaxation losses. We found the classical Half-Gauss shape<sup>15</sup> to be well-suited in many cases. For very small <sup>15</sup>N chemical shift differences (&lt; ca. 12 Hz), however, as found in
htt-exon-1, we could only achieve sufficient selectivity and sensitivity using a novel pulse shape derived using
optimal control theory,¹⁶ which we coin GOLEM (Gradient Optimized Line-width Exact Matching) (see supporting
information). The key feature of GOLEM is that the excitation profile and ¹⁵N relaxation-weighting for long pulses
(>100ms) conspire¹⁷ to yield a sharper excitation transition band. The improved ¹⁵N CSSF results in a satisfying
overall ¹⁵N selectivity of SNIPER that can go as low as 6-8 Hz. This is far superior to what was achieved
previously for 1D selective R₁p experiments.⁷ Another approach that reported improved ¹⁵N selectivity is the
F₁F₂-selective zz-exchange experiment, which combines selective HaHa with 2D acquisition to resolve close
¹⁵N chemical shifts.¹⁸ However, this results in profoundly longer experimental times than SNIPER and was
unsuccessful in resolving the zz-exchange cross-peaks of Figure 3a (see supporting information). Compared to
these methods, SNIPER comes with a modest cost in sensitivity for disordered or (small) folded proteins (see
supporting information). The additional signal averaging needed to compensate for this is by far outweighed by
the reduction in total experimental time.

SNIPER delivers high-quality, high-precision atom-resolved information on conformational dynamics of
individual residues in a mere fraction of the time that standard 2D NMR approaches would need to resolve
them. In practice, the most time-efficient strategy for relaxation analysis of all residues will be to first use such
2D approaches for sparse spectral regions and then apply complementary SNIPER experiments on key
residues located in dense spectral regions. Especially for IDPs, which commonly feature very crowded spectra
or limited sample stability, SNIPER will provide rapid access to conformational dynamics that previously would
have been very challenging to collect, as demonstrated here for the htt exon-1 protein. SNIPER presents a
powerful complement to other recent selective methods designed to speed up biomolecular NMR data collection
for assignment, such as FOSY,¹⁴c but importantly does not rely on ¹³C labeling. Demonstrated here for ¹⁵N R₁,
R₂, {¹H}-¹⁵N nOe and zz-exchange measurements, SNIPER should be compatible with any type of ¹H-¹⁵N
experiment that probes conformational dynamics, such as relaxation dispersion or chemical exchange
saturation transfer experiments.⁷,¹⁹ We expect SNIPER will find wide application in protein dynamics studies,
as well as for other biomacromolecules such as RNA.

## Supporting information

The Supporting Information contains the experimental section, further technical details and simulations of the
SNIPER pulse sequence, assessment of SNIPER's signal-to-noise ratio and time-gain, experimental results on
the relaxation analysis of huntingtin exon-1, experimental results on the zz-exchange analysis of the SH3
domain of SH3GL3 and comparison with the F₁F₂-selective experiment, Bruker pulse sequence codes and
practical setup guidelines. NMR raw data, Bruker pulse sequences and pulse shapes can be downloaded from
https://doi.org/10.5281/zenodo.16489356.

## Acknowledgements

We thank Emannuelle Boll (CNRS EMR 9002) for help with the SH3 domain assignment. D.S. acknowledges
funding from the Agence Nationale de la Recherche (ANR) (ANR-20-CE29-0015-01 URANUS) and an « accueil
de talent » grant from the Métropole Européenne de Lille (PUSHUP). The 600 MHz NMR spectrometer is funded
by the Nord Region Council, CNRS, Institut Pasteur de Lille, the European Community (ERDF), the French Ministry of Research and the Université de Lille and by the CTRL CPER co-funded by the European Union with the European Regional Development Fund (ERDF), by the Hauts-de-France Regional Council (contract n°17003781), Métropole Européenne de Lille (contract n°2016_ESR_05), and French State (contract n°2017-R3-CTRL-Phase1). The IR INFRANALYTICS FR2054 is gratefully acknowledged for financial support of the 900 MHz NMR spectrometer.

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