In silico description of fluorescent probes in vivo

Fluorescent imaging in vivo has became one of the most powerful tools to follow the temporal and spatial localization of a variety of intracellular molecular events. Genetically encoded fluorescent indicators using the FRET effect are routinely used although the molecular basis regulating their functioning is not completely known. Here, the structural and dynamics properties of a commonly used FRET sensor for the second messenger cAMP based on the cAMP-binding domains of the regulatory subunit of Protein Kinase A are presented. Molecular dynamics simulations allowed pinpointing the main features of cAMP driven conformational transition and dissecting the contributions of geometric factors governing the functioning of the biosensor. Simulations suggest that, although orientational factors are not fully isotropic, they are highly dynamic making the inter-chromophore distance the dominant feature, determining the functioning of the probes. It is expected that this computer-aided methodology may state general basis for rational design strategies of fluorescent markers for in vivo imaging.