Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform

Sensors

Joshua P. King¹  Hans-Joachim Wieden¹  Dustin D. Smith¹  D. Wade Abbott² 

[1] Alberta RNA Research and Training Institute (ARRTI), University of Lethbridge, Lethbridge, AB T1K 3M4, Canada;Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada;

关键词: computational biosensor design;    molecular dynamics;    fluorescence;    rapid kinetics;    carbohydrate detection;    oligogalacturonides;   

DOI  :  10.3390/s22030948

来源: DOAJ

【 摘 要 】

Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a “Computational Identification of Non-disruptive Conjugation sites” (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein–fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.

【 授权许可】

Unknown