Rational Design of a Novel Fluorescent Biosensor for β-Lactam Antibiotics from a Class A β-Lactamase

Abstract

A rational design strategy was used to construct a sensitive "turn-on" biosensor for beta-lactam antibiotics and beta-lactamase inhibitors from a class A beta-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Omega-loop of the E166C mutant close to the active site of the beta-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Omega-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent. This process is accompanied by an increase in the fluorescence of the labeled enzyme. The fluorescence intensity of the biosensor increases with the concentration of antibiotics or inhibitors, which can detect penicillin G at concentrations as low as 50 nM in water. This approach opens a possibility for converting highly active and nonallosteric enzymes into substrate-binding proteins for biosensing purposes.

Increased Structural Flexibility at the Active Site of a Fluorophore-conjugated β-Lactamase Distinctively Impacts Its Binding toward Diverse Cephalosporin Antibiotics

“…Fluorescence Measurement-Measurement of the fluorescence scanning spectra (from 410 to 600 nm) of PenP-E166Cb and PenP-E166Cb-E168L in the presence of six cephalosporins and in the presence of cefotaxime at various concentrations were performed in the same procedure as previously published (6) and are described in detail under supplemental data.…”

Monitorización terapéutica de antibióticos: Nuevas metodologías: biosensores

“…En breve, este biosensor cambió su fluorescencia intrínseca en función de la presencia de su sustrato 26,27 (Figura 1). En detalle, los investigadores demostraron, mediante un sistema de registro continuo de la fluorescencia emitida desde una cubeta única, que la emisión del biosensor cambia en el tiempo en función de la concentración y la identidad de antibióticos beta lactámicos en el medio 28 .…”

“…En detalle, los investigadores demostraron, mediante un sistema de registro continuo de la fluorescencia emitida desde una cubeta única, que la emisión del biosensor cambia en el tiempo en función de la concentración y la identidad de antibióticos beta lactámicos en el medio 28 . Originalmente, esta tecnología fue desarrollada para la determinación de antibióticos en matrices líquidas del rubro alimentario, tales como leche o yogures 26 , de modo que parece prometedor su uso en el campo clínico. En ese contexto, nuestro laboratorio se encuentra evaluando la metodología y las variables de corrección necesarias tales como el rango dinámico, el efecto matriz, los límites de detección, y la reproducibilidad, que permitan implementar el uso de este biosensor en el proceder de rutina de la unidad de cuidados intensivos.…”

Recent progress in the construction methodology of fluorescent biosensors based on biomolecules

“…The useful methodology to convert such non-allosteric proteins to fluorescent biosensors is to introduce an environmentally sensitive fluorophore within the proximity of the ligand-binding site, though this strategy might have some drawbacks as mentioned above. But several successful examples demonstrated that such a methodology is applicable for obtaining biosensors (Chan, P. H. et al 2004;Nalbant, P. et al 2004;Chan, P. H. 2008). Morii and coworkers constructed novel biosensors for inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P 4 ] by utilizing the pleckstrin homology (PH) domain of phospholipase C (PLC)  1 and the general receptor for phosphoinositides 1 (GRP1) (Figure 4), respectively.…”