Speaker
Description
The Bacillus subtilis spore coat is an outermost proteinaceous layer that protects spores against a variety of threats, including toxic chemicals and lytic enzymes, as well as predation by unicellular and multicellular eukaryotes. The coat, organised into two major morphologically distinct layers, the outer and inner coats, is composed of at least 70 proteins. The coat assembly process represents a central objective of our research. To gain structural insights into the spore coat of B. subtilis, we have attempted to purify several recombinant coat proteins, including CotY, CotE, CotW, CotV, and CotZ. We have discovered several self-assembled structures, including two-dimensional crystals and helical fibres. X-ray free-electron lasers are opening up unique opportunities to image biological materials at high resolution.
We will present our first XFEL-related data on the determination of the structure of CotY 2D crystals. We aim, in a longer-term perspective, to develop a robust technique for the structural determination of 2D crystals by studying a model sample, the CotY coat protein, to overcome the difficulties of obtaining 3D structures of proteins that form 2D crystals, which is likely the case for these proteins. To use these 2D lattices of Cot proteins for suitable fusion with proteins of interest for various applications, their structures must be determined. Highly organized macromolecular protein assemblies are attracting increasing interest as next-generation biomaterials due to their specific structural and functional diversity, offering potential applications in biocatalysis, drug delivery, and vaccine development. Even more possibilities open up if proteins are used as scaffolds to add entirely new functions by attaching enzymes, antigens or foreign proteins. In our work, we attempted to use CotY, a B. subtilis spore coat protein, as a scaffold that, when produced in E. coli, forms 2D crystalline structures characterized by high stability. However, genetic fusion, as a method for displaying foreign proteins on the surface of CotY crystals, adversely affected the formation of this macromolecular structure. To overcome this limitation, we employed the SpyTag/SpyCatcher bioconjugation system to engineer CotY variants suitable for modular protein attachment.
Acknowledgement
This work was supported by VEGA – Grant No. 2/0016/25 from the Slovak Academy of Sciences and a Grant from the Slovak Research and Development Agency under contract APVV-22-0303 to IB.
