Some animal organs contain mineralized tissues. These so-called hard tissues are mostly deposits of calcium salts, usually in the form of calcium phosphate or calcium carbonate. Examples of this include fish otoliths and mammalian otoconia, which are found in the inner ear, and they are an essential part of the sensory system that maintains body balance. The composition of ear stones is quite well known, but the role of individual components in the nucleation and growth of these biominerals is enigmatic. It is sure that intrinsically disordered proteins (IDPs) play an important role in this aspect. They have an impact on the shape and size of otoliths. It seems probable that IDPs, with their inherent ability to phase separate, also play a role in nucleation processes. This review discusses the major theories on the mechanisms of biomineral nucleation with a focus on the importance of protein-driven liquid–liquid phase separation (LLPS). It also presents the current understanding of the role of IDPs in the formation of calcium carbonate biominerals and predicts their potential ability to drive LLPS.

Mineralization in living organisms is highly regulated by the synergistic action of many macromolecules, where distinctive proteins play the role of direct modulators of the crystal growth. The number of methods for in vitro biomineralization studies in the presence of proteins is limited due to the influence of the environment on their functionality. We focus on a counter-diffusion system in the biomineralization of calcium carbonate that is based on the diffusion of salt solutions through a gel-matrix supplemented with the protein of interest. This article discusses the background of the system, physical principles, matrices commonly in use, advantages, limitations, variations, and our own experience. Special attention is paid to the overview of already published studies that applied the counter-diffusion system as a model for investigation of the biomineralization of calcium carbonate. The system has great potential to be widely used in the analysis of biomineralization-associated proteins in vitro and the biomineralization process itself. This perspective recalls the most important theoretical basis as well as practical application of the system for specialists in various fields of biomineralization.

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