Calcium sensor proteins in health and disease and their potential use in nanomedicine

Proteins are biomolecules involved in virtually every process occurring in cells, characterized by a sequence of amino acids, which confer them peculiar structural features implying specific functions. Ca2+-sensor proteins are a family of proteins whose function is determined by structural changes occurring upon Ca2+-binding, taking part in a wide range of physiological and pathological processes, among which muscle contraction and phototransduction. Unraveling the complex machinery behind these physiological processes is fundamental not only for the sake of expanding our knowledge, but also to understand which mechanisms are altered in inherited pathological conditions, in order to develop potential therapeutic approaches that may help relieving or treating these disorders. Protein therapy is one of the most promising potential treatments, allowing for the substitution of dysfunctional protein pool with physiological variants. Here, a collection of biochemical and biophysical studies focused on the physiological and pathological aspects of some Ca2+-sensor proteins is presented, together with possible therapeutic implications in nanomedicine using CaF2 nanoparticles (NP) as protein carriers. The combination of experimental and computational techniques revealed itself a complementary and exhaustive approach, allowing for a multiscale investigation of protein structural and functional properties. Indeed, the high-resolution structural information provided by Molecular Dynamics (MD) simulations and Protein Structure Network (PSN) analysis can be proficiently integrated into the biophysical and biochemical experimental framework constituted by Circular Dichroism (CD) and fluorescence spectroscopy, Dynamic Light Scattering (DLS), Ca2+-binding assays and enzymatic assays. Such integrated approaches allowed us to discover that mutations of the Neuronal Calcium Sensor (NCS) protein Guanylate Cyclase Activating Protein 1 (GCAP1) associated to retinal dystrophies did not necessarily altered protein Ca2+-affinity, but rather that the pathological dysregulation of the target enzyme Guanylate Cyclase (GC) depended on more complex mechanisms, probably involving modified intramolecular communication. Moreover, PSN analysis of MD trajectories of GCAP1 highlighted that small conformational variations may strongly impact protein functionality, as the switch between activator and inhibiting states occurs through minor structural rearrangements subsequent to the Ca2+/Mg2+ exchange in specific binding sites. This suggested again that the investigation of intramolecular communication may be the key to clarify unknown complex mechanisms of not only of Ca2+-sensor proteins, but also of proteins belonging to different superfamilies.