Research activity of the Bio-Nanosystems Laboratory aims at exploring the structural and functional principles of molecular machines working in living organisms. These machines are mainly made of proteins, have the capability for self-assembly and can fulfill complex functions in a highly-controlled fashion. Our research interest is mainly focused on understanding the structural organization and self-assembly of bacterial flagellum, and exploitation of the acquired knowledge for applications in bio-nanotechnology.
We address the problem how to fabricate self-assembling tubular nanostructures displaying catalytic and target recognition functionalities. Flagellin is the main component of bacterial flagella which can polymerize into long filaments. The hypervariable D3 domain of flagellin, situated on the outer surface of flagellar filaments, is not required for filament formation. The concept of our work is to engineer flagellin to give it various functionalities by replacing the D3 domain with suitable foreign proteins or peptide motifs without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures of high stability.
The prototype of flagellin-based polymerizable enzymes has been created by replacing the hypervariable central portion of flagellin with the amino acid sequence of the xylanase A enzyme. A flagellin-GFP fusion construct has been also developed which exhibits intensive fluorescence and is capable of efficient filament formation. With the aim to develop flagellin-based binding proteins, fusions of flagellin with single domain antibodies (nanobodies) have been fabricated. These flagellin variants can self-assemble into tubular nanostructures displaying thousands of regularly arranged binding sites on their surface. Such functionalized flagellar nanorods may be applied to create capture layers in biosensors. Our results demonstrate that flagellin-based fusion proteins may serve as building blocks to form filamentous nanostructures. Multifunctional nanotubes obtained by directed co-polymerization of functionalized flagellin variants offer potential applications in medical diagnostics, environmental monitoring or bionanotechnology.