Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations.
Ines ReinartzMona SarterJulia TenhaefHenning HöfigMartina PohlAlexander SchugAndreas Maximilian StadlerJörg FitterPublished in: Sensors (Basel, Switzerland) (2021)
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.
Keyphrases
- monte carlo
- energy transfer
- single molecule
- quantum dots
- molecular dynamics simulations
- binding protein
- blood glucose
- molecular dynamics
- high glucose
- living cells
- diabetic rats
- high resolution
- atomic force microscopy
- systematic review
- type diabetes
- drug induced
- magnetic resonance imaging
- endothelial cells
- metabolic syndrome
- oxidative stress
- randomized controlled trial
- adipose tissue
- computed tomography
- weight loss