Investigation of conformational dynamics of single fluorescent protein-based biosensors by molecular dynamics simulations

Genetically encoded fluorescent biosensors (GEFBs) proved to be reliable tracers for manymetabolites and cellular processes. They have a modular structure where a fluorescentprotein (FP) is genetically fused to a sensing protein which undergoes a conformationalchange upon ligand binding. This change triggers a shift in the chromophore environment,altering the FP's spectral characteristics. The structural elements crucial for effectivebiosensors are only discerned retrospectively, when crystal structures of both ligand-boundand ligand-free forms become accessible. Consequently, developing new biosensors tailoredto specific analytes often involves extensive trial and error processes. In this thesis, weinvestigate the structural determinants of high performance biosensors in both ligand boundand unbound conformations, representing the bright (ON) and dark (OFF) states respectively,by using all-atom classical molecular dynamics (MD) simulations. We characterize the shiftsin hydrogen bond occupancies over the total sensor structure to shed light on the allostericmodulation of chromophore environment. Furthermore, we deduce two strong indicators fordistinguishing the ON state of high performance sensors; a continous network of hydrogenbonds from the ligand binding site up to the chromophore environment and reduced waterdensity around the chromophore. Our results form the groundwork for the efficient design ofnew biosensors by reducing experimental screening time.execution rate.