Single molecule force spectroscopy characterization of avidin-based receptors and nuclear pore proteins

Molecular interaction forces play a key role in many essential physiological processes. Gaining knowledge of such intermolecular interactions mechanisms is of fundamental importance for understanding living systems, improving medical diagnosis and treatment, and developing new biotechnologies. Atomic Force Microscopy-based Molecular Recognition Force Spectroscopy (MRFS) allows the measurement of interaction forces between two single molecules with nanometer accuracy and piconewton force sensitivity. Thus MRFS provides detailed insights into structure, dynamics and function of single molecules, as well as complex physiological mechanisms. The aims of this thesis were firstly to characterize the interaction of new receptors capable to detect testosterone on the single molecule level and, secondly, to obtain a detailed view and molecular understanding of the transport mechanism between the cytoplasm and the nucleus of a cell. Avidin-biotin technology has been used for many years in a variety of applications in many different fields of bio-science. Its biological, chemical, and physical properties, such as femto-molar affinity or stability under harsh conditions, make the avidin-biotin system also to a promising candidate for protein engineering. Using random mutagenesis in the biotin binding area of avidin and phage display as a selection tool, new receptors with the ability to bind other small ligands can be generated. Here, two recently developed steroid-binding avidin mutans called Tes29 and TesATVN were investigated. Using MRFS it was shown that both molecules are able to specifically bind testosterone in their biotin-binding pocket. Furthermore, dynamic MRFS and competitive binding experiments with testosterone and biotin imply a decreased cross-reactivity of TesATVN for biotin. The nuclear pore complex (NPC) is a huge protein assembly embedded in the nuclear membrane, which mediate the transport of molecules between the cytoplasm and the nucleus. The key players of the selective transport and the constructor of a highly-selective permeability barrier are intrinsically disordered proteins containing phenylalanine-glycine motifs (FG nups), which line the periphery and the interior of the NPC transport conduit. Their FG repeats serve as ligands for transport shuttling factors (karyopherines), which use this interaction to open and pass the barrier, thereby chaperoning macromolecules through the pore. The mechanisms of molecule translocation and the architecture of the NPC permeability barrier are still unclear. So as to elucidate structure and function of the nuclear barrier, the interaction force between two FG nups, and between karyopherines (here importin) and FG nups were measured and the molecular dissociations rates were determined. It was shown that the forces and molecular dissociation rates of two FG motifs interacting as a pair are essentially equal to the interaction between importin and one FG motif. This implies that importins can diffuse across the nuclear pore barrier by temporarily substituting FG repeat interactions of nups with energetically equivalent importin-FG repeat interactions. Furthermore, the experiments revealed that importin recognized varied FG domains differently, indicating distinct pathways in the pore channel. Additionally, importin induced an aggregation of FG nups resulting in the formation of a hydrogel shell around itself. (author)