Search

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 vitrobiomineralization 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 vitroand 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.

The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27gene promoter. The conformational changes in the protein–DNA during the formation of the UspDBD-EcRDBD-hsp27complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5′- and/or 3′-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5′-end of the regulatory element and moved close to the 3′-end. An analysis was also done on the effect that DHR38DBD, the Drosophilaortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27bending was induced by DHR38DBD. Fluorescence data revealed that hsp27had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27in the presence of EcRDBD is presented.