Aromatic 19F-13C TROSY: a background-free approach to probe biomolecular structure, function, and dynamics

Abstract

Obtaining atomic level information about the structure and dynamics of biomolecules is critical to understand 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 to large biological systems. Here, we take advantage of the high sensitivity and the broad chemical-shift range of 19 F nuclei, and leverage the remarkable relaxation properties of the aromatic 19 F- 13 C spin pair to disperse 19 F resonances in a 2-dimensional transverse relaxation optimized TROSY spectrum. We demonstrate the application of the 19 F- 13 C TROSY to investigate proteins and nucleic acids. This experiment expands the scope of 19 F NMR in the study of structure, dynamics and function of large and complex biological systems and provides a powerful background-free NMR probe.

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…Schwalbe and coworkers used the latter method to probe the effect of all 2-F-Al abeling in the 73 nt guanine sensing riboswitch from bacillus subtilis. [3a] Only four resonances of sixteen could be assigned illustrating that in larger RNAs resonance assignment issues arise.Thus,anexpansion of the 19 Fstable isotope labeling scheme to yield a 19 F- 13 C-spin topology,such as a [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-labeling pattern in pyrimidines,would be desirable to evade resonance overlap issues in larger RNAs by adding asecond 13 Cdimension. Theapplication of 19 F-NMR to study high molecular weight systems is also hampered by the fast transverse relaxation by the chemical shift anisotropy (CSA) mechanism.…”

“…In ar ecent work this issue was addressed by introducing aromatic 19 F- 13 C-spin topologies in proteins and DNA, in which the cancellation of the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms lead to avery favorable NMR spectroscopic behavior in aTROSY type experiment. [6] Here,wereport on the synthetic access to [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-uridine (U) and -cytidine (C) phosphoramidites for solid phase RNAsynthesis.W edemonstrate the favorable spectroscopic behavior of the [5-19 F, 5-13 C]pyrimidines in two model RNAs:the 61 nt human hepatitis B virus encapsidation signal epsilon (hHBV e)a nd the 59 nt pre-microRNA21. [7] Thefeasibility of the 13 C/ 19 Flabeling by solid phase RNAsynthesis is shown and used for aresonance assignment protocol based on [5-19 F, 5-13 C]/[5-19 F]-uridine labeling schemes.F or the 61 nt hHBV e RNAw eo bserved afavorable TROSY effect and compared the performance of [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 13 C]-U labels with [6-1 H, 6-13 C]-U labels.T he novel stable isotope (SI) labeling scheme will facilitate NMR studies of large RNAs and high molecular weight RNA protein complexes as it was recently demonstrated for high molecular weight proteins systems.…”

Spotlight on the Ballet of Proteins: The Structural Dynamic Properties of Proteins Illuminated by Solution NMR

“…Protein NMR strategies are readily applicable to analyze the structure and dynamics of biologics and their heterogeneities. As biologics are mostly expressed in mammalian cells, and therefore cannot be deuterated to high levels, 15 N-detected experiments with TROSY selection are an attractive option because 15 N transverse relaxation is least affected by deuteration [120][121][122], along with the recently developed 19 F- 13 C TROSY technique for aromatic sidechains [123]. In addition, medium-sized molecules such as cyclic peptides and macrocycles tend to be flexible in solution.…”

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…Forschungsartikel phosphoramidites 7 and 11 were available RNAs with sizes between 30 and 60 nts were synthesized. First, the 30 nt HIV TAR-2 with six uridines and the 61 nt hHBV e RNAw ith eighteen uridines were synthesized and six and five [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-U labels were incorporated in each RNA, respectively. We obtained high quality 19 F- 13 CT ROSY spectra within as hort time on aP rodigy TCI probe with the proton coil tuned to the 19 Fr esonance frequency for 0.5 mm RNA samples.T he additional 13 Cd imension allowed to resolve the resonance overlap,w hich was earlier observed for U23, U25 and U31 in the 1D-19 Fs pectrum.…”