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Optimization of Caged Electrophiles for Improved Monitoring of Cysteine Reactivity in Living Cells.

ABSTRACT: Cysteine residues play critical roles in protein function and are susceptible to numerous post-translational modifications (PTMs) that serve to modulate the activity and localization of diverse proteins. Many of these PTMs are highly transient and labile, thus necessitating methods to study these modifications directly within the context of living cells. We previously reported a caged electrophilic probe, CBK1, that can be activated by UV for temporally controlled covalent modification of cysteine residues in living cells. To improve upon the number of cysteine residues identified in cellular cysteine-profiling studies, the reactivity and uncaging efficiency of a panel of caged electrophiles were explored. We identified an optimized caged electrophilic probe, CIK4, that affords significantly improved coverage of cellular cysteine residues. The broader proteome coverage afforded by CIK4 renders it a useful tool for the biological investigation of cysteine-reactivity changes and PTMs directly within living cells and highlights design elements that are critical to optimizing photoactivatable chemical probes for cellular labeling.

SUBMITTER: Abo M

PROVIDER: S-EPMC5209257 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Optimization of Caged Electrophiles for Improved Monitoring of Cysteine Reactivity in Living Cells.

Abo Masahiro M Bak Daniel W DW Weerapana Eranthie E

Chembiochem : a European journal of chemical biology 20161205 1

Cysteine residues play critical roles in protein function and are susceptible to numerous post-translational modifications (PTMs) that serve to modulate the activity and localization of diverse proteins. Many of these PTMs are highly transient and labile, thus necessitating methods to study these modifications directly within the context of living cells. We previously reported a caged electrophilic probe, CBK1, that can be activated by UV for temporally controlled covalent modification of cystei ...[more]

PMID: 27813293

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Project description:1.2,2'-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups.

Dataset Information

Optimization of Caged Electrophiles for Improved Monitoring of Cysteine Reactivity in Living Cells.

Publications

Optimization of Caged Electrophiles for Improved Monitoring of Cysteine Reactivity in Living Cells.

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