Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 µM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 µM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

[Biomineralization--precision of shape, structure and properties controlled by proteins]. / Biomineralizacja--kontrolowana przez bialka precyzja ksztaltu, struktury i wlasciwosci.

ABSTRACT Biomineralization is the process of the formation of crystal structures that is under biological control. Living organisms produce structures such as bone, teeth, otoliths, otoconia or shells. Although the chemical composition of these tissues is similar to corresponding inorganic minerals, their structure and mechanical properties differ significantly. This may be because of how they are adapted for the functions they perform. The precise control of the formation of biominerals starting with the early nucleation stage influences how the final tissues are formed. The key factors which determine the size, shape, internal structure and properties of biominerals are proteins which control the nucleation and growth of the crystals. Biomineralization is a multi-step process involving protein-protein interactions, as well as interactions between proteins and inorganic fraction. Due to their specific properties, intrinsically disordered proteins (IDPs) perform a particularly important role in the control of the biomineralization process. This article contains an overview of biominerals that are naturally occurring and describes the structures and mineralization mechanisms of the most important of them. The main part of this work was dedicated to the role of proteins which control crystal growth.