Production and characterisation of protein crystals in hydrogels to support inorganic precipitation in confined spaces

Abstract

The present Doctoral Thesis manuscript is the result of the scientific work carried out by Mariia Savchenko during her doctoral studies. The thesis is focused on crystallization processes: from magnetite biominerallization to crystallization of protein macromolecules. The thesis is organized in the following structure: a general introduction, two chapters and the general conclusions. Each chapter has a brief focus introduction, the main objectives, experimental section, results and discussion, and ends with the conclusions. The Introduction contextualizes the work. It describes the fundamental of nucleation and crystal growth theories; factors that modify these processes: environment (gels and confinement media), external stimulus (ultrasonic waves); and briefly describes the substances used in the thesis. In Chapter 1 entitled “Lysozyme crystallisation in hydrogel media under ultrasound irradiation” tells how ultrasonic waves affect the protein nucleation and growth in a hydrogel media. As first approach in this project, the specific set-up was designed, and the media that allow studying the effect without any interruptions were characterized. We showed that the application of ultrasound waves of selected energy affects the crystallization behaviour of lysozyme resulting in an induction of the nucleation and therefore affecting the final crystal size. These effect was observed in solution and in agarose if the concentration is below 0.100 (w/v) %. We propose this eco-friendly source of energy to control the production of protein crystals and to set desirable parameters. In Chapter 2 entitled “Protein crystals as a template for in situ formation of magnetite nanoparticles”, protein crystals were used as a reaction vessel to study the crystallization of another compound — magnetite, in confined spaces. The project was inspired by the magnetosomes of magnetotactic bacteria which produce magnetite with unusual morphologies, homogeneous size and superparamagnetic properties. In our case, the pores of the protein crystals control the formation of magnetite. We obtained homogeneous nanoparticles of 2 nm size regardless time, dimension of protein channel and crystalline/amorphous state. From the three model proteins used, maturation to magnetite nanoparticle was observed only in one case. The manuscript ends with the scientific publications supporting the work.