Abstract
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.
Highlights
Introduction published maps and institutional affilBiosensors are analytical devices that use biological components to detect target molecules and produce a measurable output
The scoring algorithm used for the current study focuses entirely on changes in backbone dihedral angle dynamics, and data were examined for the apo vs. various different ligand-bound states of TogB
TogB apo, TogB-unsatdigalUA, TogB-digalacturonic acid (digalUA), and TogB-trigalUA were each subjected to 100 ns molecular dynamics simulations in triplicate and analyzed using the Computational Identification of Non-disruptive Conjugation sites” (CINC) pipeline to determine amino acid FScore2.0
Summary
Introduction published maps and institutional affilBiosensors are analytical devices that use biological components to detect target molecules and produce a measurable output. A popular approach to biosensor design is the conjugation of a fluorescent group to a solute-binding protein that harbors specificity for the target molecule, exploiting ligand-induced fluorescence changes in the protein–fluorophore conjugate to detect a target [1,2]. These protein-based biosensors have been utilized for amino acid detection [3,4,5,6], anion or cation detection [4,7,8,9,10], nucleotide detection [11,12,13], and carbohydrate detection [4,14,15,16,17,18,19]. Further innovation is required to move beyond these iations
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