Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations

Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein.

Glucose-Binding of Periplasmic Protein GltB Activates GtrS-GltR Two-Component System in Pseudomonas aeruginosa

A two-component system GtrS-GltR is required for glucose transport activity in P. aeruginosa and plays a key role during P. aeruginosa-host interactions. However, the mechanism of action of GtrS-GltR has not been definitively established. Here, we show that gltB, which encodes a periplasmic glucose binding protein, is essential for the glucose-induced activation of GtrS-GltR in P. aeruginosa. We determined that GltB is capable of binding to membrane regulatory proteins including GtrS, the sensor kinase of the GtrS-GltR TCS. We observed that alanine substitution of glucose-binding residues abolishes the ability of GltB to promote the activation of GtrS-GltR. Importantly, like the gtrS deletion mutant, gltB deletion mutant showed attenuated virulence in both Drosophila melanogaster and mouse models of infection. In addition, using CHIP-seq experiments, we showed that the promoter of gltB is the major in vivo target of GltR. Collectively, these data suggest that periplasmic binding protein GltB and GtrS-GltR TCS form a complex regulatory circuit that regulates the virulence of P. aeruginosa in response to glucose.

Study of Glucose Binding Protein Encapsulated Gold Nanoclusters by Molecular Dynamic Simulation

Protein encapsulated gold nanoclusters has attracted great attention for their excellent fluorescent properties and potential biomedical applications. Glucose Binding Protein (GBP) has a high sensitivity and selectivity to glucose binding that makes them ideal for biosensor development. It is anticipated that GBP encapsulated gold nanoclusters could be a promising glucose sensor. Here we investigated the growth of gold nanoclusters in GBP using Molecular Dynamics (MD) simulation. To facilitation the nucleation of gold nanoclusters at specific sites, cysteine mutations were introduced in GBP. It is found that the nucleation site of gold nanoclusters inside mutant GBP are different from those in native GBP. Gold nanoclusters were formed near the mutated cysteine and tyrosine residues. Glucose remained in the binding site of a mutant GBP with gold nanoclusters although no conformational change was observed in MD simulation, similar to a native GBP. This work suggests the possibility of growing gold nanoclusters in the designed site within GBP and a new glucose sensor based on mutated GBP protected gold nanoclusters.