A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

Abstract

Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions. Graphic abstract

The ribosome stabilizes partially folded intermediates of a nascent multi-domain protein

“…The 2D 1 H, 15 N-SOFAST HMQC experiments 46 were recorded with acquisition times of 50.4 and 29.5 ms in the direct and indirect dimensions, respectively, and a recycle delay of 100 ms. The 19 F CEST measurements 36 47 were recorded using different spin-lock times with a spin-lock field of 7,500 Hz and the 19 F frequency carrier centred at −62.6 ppm (isolated) or −62.2 ppm (RNC).…”

“…2D 1 H, 15 N-SOFAST HMQC experiments 46 were recorded with acquisition times of 50.4 and 29.5 ms in the direct and indirect dimensions respectively, and a recycle delay of 100 ms. 19 F CEST measurements 36 were recorded with an acquisition time of 200 ms and a recycle delay of 30 ms, weak B 1 field of 15 Hz applied for a saturation time of 800 ms at saturation frequencies of either -40, -61.2 and -61.3 ppm, or -40, -62.2, -61.8 and -62.6 ppm. 19 F on-resonance R 1ρ measurements 47 were recorded using different spin-lock times with a spin-lock field of 7500 Hz, and the 19 F frequency carrier centred at -62.6 (isolated) or -62.2 ppm (RNC).…”

Fluorine NMR study of proline-rich sequences using fluoroprolines

“…The comparison of transverse relaxation at two different effective B 1 fields revealed the presence of motions occurring at the microsecond to millisecond timescales. It should be noted that the presence of many 1 H-19 F couplings within the FPro spin system implies that recently developed 19 F relaxation dispersion experiments cannot be applied (Overbeck et al, 2020). The dispersion of proton frequencies in the fluorinated prolines enable their selective excitation, a feature that was exploited for the selective Hγ -Fγ NOE and can be further used to develop sequences adapted to FPro spin systems.…”

Fluorine NMR study of proline-rich sequences using fluoroprolines

“…The comparison of transverse relaxation at two different effective B 1 fields revealed the presence of motions occurring at the µs to ms time-scale. It should be noted that the presence of many 1 H-19 F couplings within the FPro spin system implies that recently developed 19 F relaxation dispersion experiments cannot be applied (Overbeck et al, 2020). The dispersion of proton 420 frequencies in the fluorinated prolines enable their selective excitation, a feature that was exploited for the selective Hγ-Fγ NOE and can be further used to develop sequences adapted to FPro spin systems.…”