CaF2 nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies

Abstract

CaF₂-based nanoparticles (NP) are promising biocompatible tools for nanomedicine applications. The structure of the NP crystal lattice allows for specific interactions with Ca²⁺-binding proteins through their EF-hand cation binding motifs. Here we investigated the interaction of 23 nm citrate-coated CaF₂ NP with a calcium sensor protein GCAP1 that is normally expressed in photoreceptor cells and involved in the regulation of the early steps of vision. Protein–NP interactions were thoroughly investigated for the wild type (WT) GCAP1 as well as for a variant carrying the Asp 100 to Glu mutation (D100E), which prevents the binding of Ca²⁺ to the highest affinity site and is linked to cone dystrophy. Circular dichroism and fluorescence spectroscopy showed that protein structure and Ca²⁺-sensing capability are conserved for both variants upon interaction with the NP surface, although the interaction mode depends on the specific occupation of Ca²⁺-binding sites. NP binding stabilizes the structure of the bound GCAP1 and occurs with nanomolar affinity, as probed by isothermal titration calorimetry. Surface plasmon resonance revealed a fully reversible binding compatible with physiologically relevant kinetics of protein release whereas biochemical assays indicated a residual capability for NP-dissociated GCAP1 to regulate the target retinal guanylate cyclase. Our study constitutes a proof of concept that CaF₂ NP could be optimized to serve as biologically compatible carriers of high amounts of functional GCAP1 in photoreceptors affected by retinal dystrophies.