Ranking Single Fluorescent Protein-Based Calcium Biosensor Performance by Molecular Dynamics Simulations

Genetically encoded fluorescent biosensors (GEFBs) have become indispensable tools for visualizing biological processes <i>in vivo.</i> A typical GEFB is composed of a sensory domain (SD) that undergoes a conformational change upon ligand binding or enzymatic reaction; the SD is genetically fused with a fluorescent protein (FP). The changes in the SD allosterically modulate the chromophore environment whose spectral properties are changed. Single fluorescent (FP)-based biosensors, a subclass of GEFBs, offer a simple experimental setup; they are easy to produce in living cells, structurally stable, and simple to use due to their single-wavelength operation. However, they pose a significant challenge for structure optimization, especially concerning the length and residue content of linkers between the FP and SD, which affect how well the chromophore responds to conformational change in the SD. In this work, we use all-atom molecular dynamics simulations to analyze the dynamic properties of a series of calmodulin-based calcium biosensors, all with different FP-SD interaction interfaces and varying degrees of calcium binding-dependent fluorescence change. Our results indicate that biosensor performance can be predicted based on distribution of water molecules around the chromophore and shifts in hydrogen bond occupancies between the ligand-bound and ligand-free sensor structures.