Design of De Novo Mini Fluorescence Activating Proteins as pH and Calcium Biosensors

Abstract

Recent advances in de novo protein design have enabled the design of mini fluorescence activating proteins (mFAPs): mini β-barrel topologies that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. mFAPs offer advantages over conventional fluorescent proteins, such as smaller molecular size, smaller genetic footprint, and high signal-to-noise ratio. As described herein, I employ structure-guided protein design to engineer mFAPs to be brighter (only 3.5-fold dimmer than enhanced green fluorescent protein (EGFP)), and both selective and promiscuous toward different chromophores (DFHBI, DFHBI-1T, and DFHO), revealing additional advantages of spectral tunability, external spatiotemporal control over fluorescence, and photostability. Additionally, we engineered mFAPs to have stable folds at low pH, and to bind the protonated (phenolic) and deprotonated (phenolate) states of DFHBI, conferring pH-responsive fluorescence based on the protonation/deprotonation equilibrium of DFHBI with a ~250-fold change in ratiometric fluorescence from pH 3.6-8.4. Furthermore, molecular engineering of mFAPs to exhibit Ca2+-responsive fluorescence was achieved by grafting one, two, or four EF-hand motifs onto mFAP loops juxtaposing the DFHBI binding pocket to generate fluorescent Ca2+ biosensors capable of monitoring Ca2+ transients in cyto with high photostability. Both positive allosteric coupling and negative allosteric coupling between Ca2+ and DFHBI binding is observed, with >500-fold changes in the thermodynamic dissociation constant for Ca2+ depending on the number of EF-hand motifs used. Structural insight into the positive allosteric modulation mechanism is provided via X-ray crystallographic data and molecular dynamics simulations. While mFAPs still have much room for improvement, it is anticipated that these already improved mFAPs have numerous advanced imaging applications within the fields of molecular biology, nanobiotechnology, and beyond.