Garima Ghale

• This thesis describes the application of systems composed of a fluorophore and macrocyclic host as a sensitive and versatile analytical method to study biological processes, in particular, transport through the lipid-bilayer biomembrane and reactions catalyzed by proteases. While it is acknowledged that only small and hydrophobic molecules can pass through the bilayer directly and while the importance of channel proteins for selective uptake is well recognized, methods to directly and sensitively monitor these membrane transport processes remain in high demand. In the first part of the thesis, I introduce an in vitro fluorescence-based biomembrane assay—the supramolecular tandem membrane assay—based on host–guest, macrocycle–fluorophore reporter pairs encapsulated inside liposomes. Three features make this novel method unique. First, the tandem membrane assay allows label-free continuous monitoring of translocation through the lipid bilayer or through channel-proteins, which was previously not possible. Second, the assay is economic and versatile, i.e., applicable to both, diffusion through the membrane and translocation through channel proteins of different classes of translocating biomolecules. Third, the assay allows direct access to kinetic information of analyte permeation on microsecond-to-hours time scales. As a first application of the tandem membrane assay, we applied it to the unsolved biological question, whether cationic antimicrobial peptides, in particular, protamine, can enter into the cell through an outer membrane channel protein. For the first time, conclusive experimental evidence has been provided that such antimicrobial peptides can indeed enter through bacterial channels. Protease assays for natural or unlabelled synthetic substrates are very much sought after, but the few assays that have been developed are limited to the general detection of protease activity. In the second part of this thesis, I present the label-free fluorescence-based protease assay—the supramolecular tandem protease assay. This newly developed label-free method overcomes the use of covalently attached fluorescent labels in peptides by, again, employing macrocycle–fluorophore reporter pairs. The beauty of our sensitive and versatile protease assay lies not only in being able to (i) monitor protease activity in real-time, but also in its utility to (ii) profile the substrate specificity of a protease, (iii) to sense enzyme substrate stereoselectivity, and (iv) in its utility for rapid and convenient screening of inhibitors and protease–resistant peptides. Additionally, the tandem protease assay allows stepwise proteolytic degradation of an entire peptide to be easily followed as sequential changes of the fluorescence response, which led us to the idea of peptide fingerprinting through unique fluorescence profiles.