Probing Peptidylprolyl Bond cis/trans Status Using Distal 19F NMR Reporters

Killoran, Patrick M.; Hanson, George S M; Verhoork, Sanne J. M.; Smith, M. Scott; Gobbo, Davide Del; Lian, Lu‐Yun; Coxon, Christopher R.

doi:10.1002/chem.202203017

Cited by 2 publications

(1 citation statement)

References 43 publications

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“…Raines observed similar 19 F NMR data for Ac-Dfp-OMe in CDCl 3 and in D 2 O (Δδ FF ( trans ) 0.4–4.0 ppm, Δδ FF ( cis ) = 4.6–8.5 ppm) . These data suggested the possibility of using 4,4-difluoroproline as a probe of proline cis – trans isomerization in peptides and proteins, analogous to using Δδ βγ , but with the greater sensitivity and simplified spectra of 19 F NMR compared to 13 C NMR. − …”

Section: Introductionmentioning

confidence: 64%

4,4-Difluoroproline as a Unique ¹⁹F NMR Probe of Proline Conformation

Ganguly,

Ludwig,

Tressler

et al. 2024

Biochemistry

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Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19 F NMR-based probe of proline conformational biases and cis−trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (Δδ FF = 0−3 ppm) when a trans X−Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (Δδ FF = 5−12 ppm) difference in chemical shift in a cis X−Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that Δδ FF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudoaxial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudoequatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis−trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis−trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis−trans isomerization, protein folding, and local order in intrinsically disordered proteins.

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Section: Introductionmentioning

confidence: 64%

4,4-Difluoroproline as a Unique ¹⁹F NMR Probe of Proline Conformation

Ganguly,

Ludwig,

Tressler

et al. 2024

Biochemistry

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Proline cis/trans Isomerization in Intrinsically Disordered Proteins and Peptides

Sebák,

Szolomájer,

Papp

et al. 2023

Front. Biosci. (Landmark Ed)

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Background: Intrinsically disordered proteins and protein regions (IDPs/IDRs) are important in diverse biological processes. Lacking a stable secondary structure, they display an ensemble of conformations. One factor contributing to this conformational heterogeneity is the proline cis/trans isomerization. The knowledge and value of a given cis/trans proline ratio are paramount, as the different conformational states can be responsible for different biological functions. Nuclear Magnetic Resonance (NMR) spectroscopy is the only method to characterize the two co-existing isomers on an atomic level, and only a few works report on these data. Methods: After collecting the available experimental literature findings, we conducted a statistical analysis regarding the influence of the neighboring amino acid types (i ± 4 regions) on forming a cis-Pro isomer. Based on this, several regularities were formulated. NMR spectroscopy was then used to define the cis-Pro content on model peptides and desired point mutations. Results: Analysis of NMR spectra prove the dependence of the cis-Pro content on the type of the neighboring amino acid—with special attention on aromatic and positively charged sidechains. Conclusions: Our results may benefit the design of protein regions with a given cis-Pro content, and contribute to a better understanding of the roles and functions of IDPs.

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Probing Peptidylprolyl Bond cis/trans Status Using Distal ¹⁹F NMR Reporters

Abstract: Supporting information for this article is given via a link at the end of the document.

Cited by 2 publications

References 43 publications

4,4-Difluoroproline as a Unique ¹⁹F NMR Probe of Proline Conformation

4,4-Difluoroproline as a Unique ¹⁹F NMR Probe of Proline Conformation

Proline cis/trans Isomerization in Intrinsically Disordered Proteins and Peptides

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Probing Peptidylprolyl Bond cis/trans Status Using Distal 19F NMR Reporters

Abstract: Supporting information for this article is given via a link at the end of the document.

Cited by 2 publications

References 43 publications

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation

Proline cis/trans Isomerization in Intrinsically Disordered Proteins and Peptides

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Product

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Probing Peptidylprolyl Bond cis/trans Status Using Distal ¹⁹F NMR Reporters

4,4-Difluoroproline as a Unique ¹⁹F NMR Probe of Proline Conformation

4,4-Difluoroproline as a Unique ¹⁹F NMR Probe of Proline Conformation