Side‐chain thioamides as fluorescence quenching probes

Robkis, D. Miklos; Hoang, Eileen M.; Po, Pengse; Deutsch, Carol; Petersson, E. James

doi:10.1002/bip.23384

Cited by 10 publications

(11 citation statements)

References 28 publications

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“…Predicted E FRET values from simulations of the Cnf–Pro n –Lys(Ac S ) system compared to experimental values . The efficacy of each sampling approach (+ Chi-Sampling, + Internal cisPro, + Terminal cisPro, + CH−π Interaction) was assessed for all the three methods of computing E FRET , (a) PDA, isotopic; (b) PDA, explicit; and (c) TrESP, to determine the most accurate sampling method.…”

Section: Resultsmentioning

confidence: 99%

“…All polyproline simulations used the polyproline type-II helix (PPII) as a starting structure, where the number of prolines varied from 2 to 10 for thioleucine (Leu S ) (where the superscript S denotes the thioamide) and 2 to 6 for Lys(Ac S ). 24,25,31 To match prior experiments, the backbone thioamide-containing polyproline structures contained Cnf/Tyr/Trp at the N-terminus and the Leu S at the C-terminus, while side-chain thioamide-containing polyproline structures contained Lys(Ac S ) at the N-terminus and Cnf/Trp at the C-terminus. 24,25,31 Previously, backrub simulations have been shown to produce structure ensembles that mimic solution-phase dynamics by sampling local motions via rotations around axes defined by two backbone atoms.…”

Section: ■ Methodsmentioning

confidence: 99%

“…24,25,31 To match prior experiments, the backbone thioamide-containing polyproline structures contained Cnf/Tyr/Trp at the N-terminus and the Leu S at the C-terminus, while side-chain thioamide-containing polyproline structures contained Lys(Ac S ) at the N-terminus and Cnf/Trp at the C-terminus. 24,25,31 Previously, backrub simulations have been shown to produce structure ensembles that mimic solution-phase dynamics by sampling local motions via rotations around axes defined by two backbone atoms. 32,33 Using a modified backrub-based sampling method implemented in PyRosetta, 1000 unique backbone and side-chain conformations were generated via torsional sampling.…”

Section: ■ Methodsmentioning

confidence: 99%

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Improved Modeling of Thioamide FRET Quenching by Including Conformational Restriction and Coulomb Coupling

2020

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Thioamide-containing amino acids have been shown to quench a wide range of fluorophores through distinct mechanisms. Here, we quantitatively analyze the mechanism through which the thioamide functional group quenches the fluorescence of p-cyanophenylalanine (Cnf), tyrosine (Tyr), and tryptophan (Trp). By comparing PyRosetta simulations to published experiments performed on polyproline ruler peptides, we corroborate previous findings that both Cnf and Tyr quenching occurs via Förster resonance energy transfer (FRET), while Trp quenching occurs through an alternate mechanism such as Dexter transfer. Additionally, optimization of the peptide sampling scheme and comparison of thioamides attached to the peptide backbone and side chain revealed that the significant conformational restriction associated with the thioamide moiety results in a high sensitivity of the apparent FRET efficiency to underlying conformational differences. Moreover, by computing FRET efficiencies from structural models using a variety of approaches, we find that quantitative accuracy in the role of Coulomb coupling is required to explain contributions to the observed quenching efficiency from individual structures on a detailed level. Last, we demonstrate that these additional considerations improve our ability to predict thioamide quenching efficiencies observed during binding of thioamide-labeled peptides to fluorophore-labeled variants of calmodulin.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

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Improved Modeling of Thioamide FRET Quenching by Including Conformational Restriction and Coulomb Coupling

2020

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“…For H3K9thioac, the presence of a thioamide moiety interfered with the determining concentration by absorbance at 280 nm. Instead, concentration was determined by measuring absorbance at 260 nm using a previously reported extinction coefficient for thioacetyllysine at this wavelength 65 …”

Section: Methodsmentioning

confidence: 99%

Evaluation of acyllysine isostere interactions with the aromatic pocket of the AF9 YEATS domain

et al. 2022

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AmideÀπ interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amideÀπ interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amideÀπ interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostereÀπ interaction. We show that compared to the amideÀπ interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea-containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post-translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.

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“…Thioamides have been shown to be compatible in several protein secondary structure motifs, which allows for the exploration of hydrogen-bonding effects to and from the backbone. − Thioamides have also been useful in the study of other noncovalent interactions such as the n → π* interaction between neighboring peptide CO groups. − The different properties of thioamides and amides have also allowed thioamides to serve as minimal probes for the study of larger peptides and proteins. For instance, thioamides have been implemented as photoswitchable units to effect cis / trans isomerization of the thioamide unit. , Likewise, the thioamide can act as a quencher of fluorescent dyes to study peptide conformation and proteolysis. − In general, thioamides are viewed as subtle single-atom substitutions that provide an analytical handle for a variety of biochemical and biophysical techniques and therefore suggests their indispensable role in peptide sciences.…”

mentioning

confidence: 99%

Hydrogen Bond and Geometry Effects of Thioamide Backbone Modifications

Lampkin

VanVeller

2021

J. Org. Chem.

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Thioamide substitution of backbone peptide bonds can probe interactions along the main chain of proteins. Despite theoretical predictions of the enhanced hydrogen bonding propensities of thioamides, previous studies often do not consider the geometric constraints imposed by folded peptide secondary structure. This work addresses drawbacks in previous studies that ignored the geometry dependence and local dielectric properties of thioamide hydrogen bonding and identifies cases where thioamides may be either stronger or weaker hydrogen-bonding partners than amides.

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Side‐chain thioamides as fluorescence quenching probes

Cited by 10 publications

References 28 publications

Improved Modeling of Thioamide FRET Quenching by Including Conformational Restriction and Coulomb Coupling

Improved Modeling of Thioamide FRET Quenching by Including Conformational Restriction and Coulomb Coupling

Evaluation of acyllysine isostere interactions with the aromatic pocket of the AF9 YEATS domain

Hydrogen Bond and Geometry Effects of Thioamide Backbone Modifications

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