Research

Translocation through the bacterial membrane is an essential step in biogenesis of secretory proteins, as well as the proteins targeted to the cell envelope. Within the dedicated general secretory pathway, the proteins in form of unfolded polypeptide chains are transported through the membrane-embedded channel SecYEG with help of the motor protein SecA. Studies over the last decade has highlighted the novel roles of the accessory proteins and also membrane lipids in activity of the SecA:SecYEG machinery. In our research, we investigate:

-  the role of the lipid membrane structure and composition on protein translocation

-  the effect of periplasmic chaperones on translocation of bacterial virulence factors

SecYEG/Sec61 translocon is a universally conserved hub for protein translocation (see above) and integration of the nascent membrane proteins. The assembly of the translocon with the ribosome at the membrane interface is the major point of our interest. We employ biophysical and structural methods to elucidate the structure and dynamics of the complex along the membrane protein biogenesis.

Integral membrane proteins occupy from 30% up to 80% of membrane surface area and generate highly crowded and anisotropic environment. Despite this natural complexity, existing experimental systems largely address membrane-associated reactions either in non-physiological detergent-solubilized state, or within over-simplified minimalistic liposomes. Three directions are followed within the project:

• development of de novo protein:membrane systems, which mimic the organization of biological membranes with a focus on macromolecular crowding;
• development of genetically-encoded sensors to characterize macromolecular crowding at the membrane interfaces in vitro and in vivo;
• studying the effects of the macromolecular crowding on the functionality of the Sec machinery. Here, the Sec machinery serves as a well-characterized model system to probe protein:protein interactions and its functionality in the native-like environment.

The comprehensive analysis in designed complex environments builds a novel framework for understanding membrane protein biogenesis and will serve to bridge biochemical studies in simplified systems and in vivo folding analysis in cell biology. Developed and tailored crowded membranes will be readily available for studying diverse membrane-associated processes.

Biofilms, i.e. large aggregated bacterial communities, belong to the most abundant forms of life on Earth. The biofilms ensure survival of bacteria under severe conditions, provide the environment for the horizontal transfer of genomic information and quorum sensing, and facilitate antibiotic resistance of the opportunistic human pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus. Large exopolysaccharide molecules (EPS) universally contribute to the assembly of the biofilm matrix, but their biogenesis/secretion pathways have been barely described. In our group, we employ biochemical, biophysical and structural methods to provide a comprehensive view on the molecular mechanisms of EPS secretion.