Thioamide-Based Fluorescent Protease Sensors

Goldberg, Jacob M.; Chen, Xing; Meinhardt, Nataline; Greenbaum, Doron C.; Petersson, E. James

doi:10.1021/ja412297x

Cited by 47 publications

(45 citation statements)

References 66 publications

(110 reference statements)

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“…Our laboratory has previously developed a thioamide‐based fluorescent protease sensor system which we envisioned using to better understand the effects of thioamides on proteolysis . This system, based on our extensive studies of thioamides as fluorescence quenchers, involved placing a thioamide and a fluorophore on opposite sides of the scissile bond, so that a fluorescence turn‐on would be observed once proteolysis occurs.…”

Section: Figurementioning

confidence: 99%

Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases

et al. 2019

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Thioamide substitutions of the peptide backbone have been shown to reduce proteolytic degradation, and this property can be used to generate competitive protease inhibitors and to stabilize peptides toward degradation in vivo. Here, we present a straightforward sensor design that allows a systematic study of the positional effects of thioamide substitution by using real‐time fluorescence. Thioamide scanning in peptide substrates of five papain family cysteine proteases demonstrates that a thioamide at or near the scissile bond can slow proteolysis in all cases, but that the magnitude of the effects varies with position and protease in spite of high sequence homology. Mechanistic investigation of papain proteolysis reveals that the thioamide effects derive from reductions in both affinity (KM) and turnover number (kcat). Computational modeling allows these effects to be understood based on disruption of key enzyme–substrate hydrogen bonds, providing a model for future rational use of thioamides to confer cysteine protease resistance.

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Section: Figurementioning

confidence: 99%

Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases

et al. 2019

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“…The peptide sequence was chosen as a protease substrate based on previous studies in our laboratory so that cleavage should result in a decrease in FRET. [18] Cleavage by the proteases trypsin and cathepsin B was tested by exciting at 325 nm and monitoring emission at 380 nm (Mcm) and either 480 nm (Acd) or 530 nm (Aad). After confirming that trypsin proteolysis proceeded at a reasonable rate in a multi-well plate assay (see ESI, Figs.…”

Section: Results and Discussion: Synthesis Of Aminoacridonylalanine Fmentioning

confidence: 99%

Improving the fluorescent probe acridonylalanine through a combination of theory and experiment

Sungwienwong

Ferrie

Jun

et al. 2018

J of Physical Organic Chem

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Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluorescence spectroscopy, but it can potentially be improved by being made longer wavelength or brighter. Here, we report the synthesis of Acd core derivatives and their photophysical characterization. We also performed ab initio calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior FRET acceptor to Acd in a peptide cleavage assay, and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives which can be used in peptides and proteins.

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“…The same approach was extended to register the Trp and Tyr quenching in a distance‐dependent manner . Pro‐fluorescent peptides based on a thionamidated ‐Leu‐Leu‐ unit and a synthetic fluorophoric amino acid (7‐methoxycumarin‐4‐yl‐alanine) were shown to become fluorescent after an appropriate proteolytic attack . An intramolecular FRET study was carried out on a Phe(CN) and thioamide double‐labeled semi ‐synthetic protein, prepared via a combination of unnatural amino acid mutagenesis and native chemical ligation .…”

Section: Resultsmentioning

confidence: 99%

Endothioxopeptides: A conformational overview

et al. 2016

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Although thionamides would have been first prepared two centuries ago and their chemical and spectroscopic properties extensively investigated, only much more recently (since about 1985) a well deserved but still insufficient attention has been paid to their endothioxopeptide subfamily which nonetheless currently represents a rapidly emerging area of great scientific interest in the broader field of foldameric compounds based on biologically relevant building blocks. After two brief sections offering information on the unfortunately still limited number of endothioxopeptides discovered from natural sources but also on the impressive advancements registered in the last few years in their synthetic methods, this review article outlines the results of a detailed literature survey on the ongoing great, but not systematic, progress related to the conformational consequences generated by incorporating one (or more) thionamide group(s) into a polypeptide chain. Finally, a short discussion of the growing, but still in its infancy, class of the endoselenoxopeptide congeners is also presented.

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Thioamide-Based Fluorescent Protease Sensors

Cited by 47 publications

References 66 publications

Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases

Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases

Improving the fluorescent probe acridonylalanine through a combination of theory and experiment

Endothioxopeptides: A conformational overview

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