Limits...
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.

Show MeSH

Related in: MedlinePlus

Molecular models of the fluorescein-labelled TEM-1 V216C mutant in the free-enzyme (E) and ES* states(A) Without penicillin G, the fluorescein molecule (orange) lies close to the active site of the enzyme. (B) With penicillin G, the fluorescein molecule (orange) stays away from the active site when penicillin G (yellow) binds to Ser70 in the active site. The ribbon diagrams represent the structure of the TEM-1 V216C mutant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4155835&req=5

Figure 6: Molecular models of the fluorescein-labelled TEM-1 V216C mutant in the free-enzyme (E) and ES* states(A) Without penicillin G, the fluorescein molecule (orange) lies close to the active site of the enzyme. (B) With penicillin G, the fluorescein molecule (orange) stays away from the active site when penicillin G (yellow) binds to Ser70 in the active site. The ribbon diagrams represent the structure of the TEM-1 V216C mutant.

Mentions: In order to examine the effect of drug binding at the active site on the conformation of the attached fluorescein molecule, we conducted molecular modelling on the free enzyme (E) and ES* states of the labelled V216C mutant with penicillin G as the substrate. Figure 6 shows the molecular models of the labelled V216C mutant with and without binding to penicillin G. In the free-enzyme state, the fluorescein molecule lies close to the active site (Figure 6A). Upon binding to penicillin G, the fluorescein molecule stays away from the active site (Figure 6B). This subtle conformational change is likely to keep the fluorescein molecule away from the residues around the active site, thus reducing their fluorescence quenching on the fluorescein molecule.


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)

Molecular models of the fluorescein-labelled TEM-1 V216C mutant in the free-enzyme (E) and ES* states(A) Without penicillin G, the fluorescein molecule (orange) lies close to the active site of the enzyme. (B) With penicillin G, the fluorescein molecule (orange) stays away from the active site when penicillin G (yellow) binds to Ser70 in the active site. The ribbon diagrams represent the structure of the TEM-1 V216C mutant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Molecular models of the fluorescein-labelled TEM-1 V216C mutant in the free-enzyme (E) and ES* states(A) Without penicillin G, the fluorescein molecule (orange) lies close to the active site of the enzyme. (B) With penicillin G, the fluorescein molecule (orange) stays away from the active site when penicillin G (yellow) binds to Ser70 in the active site. The ribbon diagrams represent the structure of the TEM-1 V216C mutant.
Mentions: In order to examine the effect of drug binding at the active site on the conformation of the attached fluorescein molecule, we conducted molecular modelling on the free enzyme (E) and ES* states of the labelled V216C mutant with penicillin G as the substrate. Figure 6 shows the molecular models of the labelled V216C mutant with and without binding to penicillin G. In the free-enzyme state, the fluorescein molecule lies close to the active site (Figure 6A). Upon binding to penicillin G, the fluorescein molecule stays away from the active site (Figure 6B). This subtle conformational change is likely to keep the fluorescein molecule away from the residues around the active site, thus reducing their fluorescence quenching on the fluorescein molecule.

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