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

Overbeck, Jan H.; Kremer, Werner; Sprangers, Remco

doi:10.1007/s10858-020-00348-4

Cited by 27 publications

(40 citation statements)

References 63 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

Fluorine NMR study of proline-rich sequences using fluoroprolines

et al. 2021

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Abstract. Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to a lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering – they introduce conformational and dynamical biases – but their use as 19F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where Cγ-fluorinated prolines with opposite configurations of the chiral Cγ centre have been introduced at two positions in distinct polyproline segments. By looking at the effects of swapping these (4R)-fluoroproline and (4S)-fluoroproline within the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19F relaxation properties, and we exploit the latter in elucidating binding kinetics to the SH3 (Src homology 3) domain.

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Section: Discussionmentioning

confidence: 99%

Fluorine NMR study of proline-rich sequences using fluoroprolines

et al. 2021

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“…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.…”

Section: Discussionmentioning

confidence: 99%

Fluorine NMR study of proline-rich sequences using fluoroprolines

Sinnaeve¹,

Bouzayene

Ottoy

et al. 2021

Preprint

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Abstract. Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering – they introduce conformational and dynamical biases – but their use as 19F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where Cγ-fluorinated prolines with opposite configurations of the chiral Cγ centre have been introduced at two postions in distinct polyproline segments. By looking at the effects of swapping these (4R)- and (4S)-4-fluoroprolines witin the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19F relaxation properties, and exploit the latter in elucidating binding kinetics to the SH3 domain.

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“…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).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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Co-translational folding is crucial to ensure the production of biologically active proteins. The ribosome can alter the folding pathways of nascent polypeptide chains, yet a structural understanding remains largely inaccessible experimentally. We have developed site-specific labelling of nascent chains to detect and measure, using 19F nuclear magnetic resonance (NMR) spectroscopy, multiple states accessed by an immunoglobulin-like domain within a tandem repeat protein during biosynthesis. By examining ribosomes arrested at different stages during translation of this common structural motif, we observe highly broadened NMR resonances attributable to two previously unidentified intermediates, which are stably populated across a wide folding transition. Using molecular dynamics simulations and corroborated by cryo-electron microscopy, we obtain models of these partially folded states, enabling experimental verification of a ribosome-binding site that contributes to their high stabilities. We thus demonstrate a mechanism by which the ribosome could thermodynamically regulate folding and other co-translational processes.

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

Cited by 27 publications

References 63 publications

Fluorine NMR study of proline-rich sequences using fluoroprolines

Fluorine NMR study of proline-rich sequences using fluoroprolines

Fluorine NMR study of proline-rich sequences using fluoroprolines

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

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