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Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening.

Cheong WL, Tsang MS, So PK, Chung WH, Leung YC, Chan PH - Biosci. Rep. (2014)

Bottom Line: The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site.The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site.Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state.

View Article: PubMed Central - PubMed

Affiliation: *State Key Laboratory of Chirosciences, Food Safety and Technology Research Centre, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, People's Republic of China.

ABSTRACT
We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening.

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Time-course fluorescence measurements on the labelled V216C mutant in the presence of trypsinThe fluorescence of the labelled V216C mutant increases in the course of trypsin digestion. The labelled V216C mutant (0.2 mg·ml−1) was mixed with trypsin (0.01 mg·ml−1) in 50 mM potassium phosphate buffer (pH 7.0).
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Figure 7: Time-course fluorescence measurements on the labelled V216C mutant in the presence of trypsinThe fluorescence of the labelled V216C mutant increases in the course of trypsin digestion. The labelled V216C mutant (0.2 mg·ml−1) was mixed with trypsin (0.01 mg·ml−1) in 50 mM potassium phosphate buffer (pH 7.0).

Mentions: We compared the fluorescence of the fluorescein molecule in the folded state of the labelled V216C mutant and its fragmented state after trypsin digestion. This comparative study can verify whether the fluorescein molecule fluoresces weakly when staying close to the active site of the enzyme (folded state) and gives stronger fluorescence when staying away from the active site (fragmented state). The digestion on the labelled V216C mutant by trypsin was first monitored by SDS/PAGE analysis. Supplementary Figure S10 (available at http://www.bioscirep.org/bsr/034/bsr034e136add.htm) shows the fluorescent image of the SDS/PAGE gel for the labelled V216C mutant before and after trypsin digestion. Before trypsin digestion, the labelled V216C mutant shows a green fluorescent band at the position corresponding to the undigested form (Figure S10). After incubation with trypsin for 8 h, some green fluorescent bands appear at lower positions, indicating that the labelled V216C mutant was cleaved into smaller peptide fragments by trypsin (Figure S10). We then examined the fluorescence of the labelled V216C mutant in response to trypsin digestion. Interestingly, the fluorescence of the labelled V216C mutant increases as a function of time during the course of trypsin digestion (Figure 7). This experimental observation implies that the fluorescein molecule is likely to experience fluorescence quenching when it stays close to the enzyme's active site in the folded state. Such fluorescence quenching is relieved when the fluorescein molecule stays away from the active site as a result of the trypsin digestion on the structure of the labelled V216C mutant, thus leading to the fluorescence enhancement of the fluorescein molecule. Taking the results of the ESI–MS, molecular modelling and trypsin digestion studies together, the fluorescein molecule is likely to lie close to the active site in the free enzyme state (E) and fluoresces weakly due to fluorescence quenching by the residues around the active site. Upon binding to β-lactam antibiotics, the fluorescein molecule is likely to stay away from the active site and experience weaker fluorescence quenching, thus giving stronger fluorescence in the substrate-bound state (ES and ES*). After enzymatic hydrolysis, the hydrolysed product (P) will be released from the active site, and therefore the fluorescein molecule will approach the active site again and restore its weak fluorescence.


Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening.

Cheong WL, Tsang MS, So PK, Chung WH, Leung YC, Chan PH - Biosci. Rep. (2014)

Time-course fluorescence measurements on the labelled V216C mutant in the presence of trypsinThe fluorescence of the labelled V216C mutant increases in the course of trypsin digestion. The labelled V216C mutant (0.2 mg·ml−1) was mixed with trypsin (0.01 mg·ml−1) in 50 mM potassium phosphate buffer (pH 7.0).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4155835&req=5

Figure 7: Time-course fluorescence measurements on the labelled V216C mutant in the presence of trypsinThe fluorescence of the labelled V216C mutant increases in the course of trypsin digestion. The labelled V216C mutant (0.2 mg·ml−1) was mixed with trypsin (0.01 mg·ml−1) in 50 mM potassium phosphate buffer (pH 7.0).
Mentions: We compared the fluorescence of the fluorescein molecule in the folded state of the labelled V216C mutant and its fragmented state after trypsin digestion. This comparative study can verify whether the fluorescein molecule fluoresces weakly when staying close to the active site of the enzyme (folded state) and gives stronger fluorescence when staying away from the active site (fragmented state). The digestion on the labelled V216C mutant by trypsin was first monitored by SDS/PAGE analysis. Supplementary Figure S10 (available at http://www.bioscirep.org/bsr/034/bsr034e136add.htm) shows the fluorescent image of the SDS/PAGE gel for the labelled V216C mutant before and after trypsin digestion. Before trypsin digestion, the labelled V216C mutant shows a green fluorescent band at the position corresponding to the undigested form (Figure S10). After incubation with trypsin for 8 h, some green fluorescent bands appear at lower positions, indicating that the labelled V216C mutant was cleaved into smaller peptide fragments by trypsin (Figure S10). We then examined the fluorescence of the labelled V216C mutant in response to trypsin digestion. Interestingly, the fluorescence of the labelled V216C mutant increases as a function of time during the course of trypsin digestion (Figure 7). This experimental observation implies that the fluorescein molecule is likely to experience fluorescence quenching when it stays close to the enzyme's active site in the folded state. Such fluorescence quenching is relieved when the fluorescein molecule stays away from the active site as a result of the trypsin digestion on the structure of the labelled V216C mutant, thus leading to the fluorescence enhancement of the fluorescein molecule. Taking the results of the ESI–MS, molecular modelling and trypsin digestion studies together, the fluorescein molecule is likely to lie close to the active site in the free enzyme state (E) and fluoresces weakly due to fluorescence quenching by the residues around the active site. Upon binding to β-lactam antibiotics, the fluorescein molecule is likely to stay away from the active site and experience weaker fluorescence quenching, thus giving stronger fluorescence in the substrate-bound state (ES and ES*). After enzymatic hydrolysis, the hydrolysed product (P) will be released from the active site, and therefore the fluorescein molecule will approach the active site again and restore its weak fluorescence.

Bottom Line: The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site.The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site.Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state.

View Article: PubMed Central - PubMed

Affiliation: *State Key Laboratory of Chirosciences, Food Safety and Technology Research Centre, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, People's Republic of China.

ABSTRACT
We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening.

Show MeSH
Related in: MedlinePlus