## Reconstitution and Membrane Topology of Mistic from Bacillus subtilis

• Cells and organelles are enclosed by membranes that consist of a lipid bilayer harboring highly diverse membrane proteins (MPs). These carry out vital functions, and α-helical MPs, in particular, are of outstanding pharmacological importance, as they comprise more than half of all drug targets. However, knowledge from MP research is limited, as MPs require membranemimetic environments to retain their native structures and functions and, thus, are not readily amenable to in vitro studies. To gain insight into vectorial functions, as in the case of channels and transporters, and into topology, which describes MP conformation and orientation in the context of a membrane, purified MPs need to be reconstituted, that is, transferred from detergent micelles into a lipid-bilayer system. The ultimate goal of this thesis was to elucidate the membrane topology of Mistic, which is an essential regulator of biofilm formation in Bacillus subtilis consisting of four α-helices. The conformational stability of Mistic has been shown to depend on the presence of a hydrophobic environment. However, Mistic is characterized by an uncommonly hydrophilic surface, and its helices are significantly shorter than transmembrane helices of canonical integral MPs. Therefore, the means by which its association with the hydrophobic interior of a lipid bilayer is accomplished is a subject of much debate. To tackle this issue, Mistic was produced and purified, reconstituted, and subjected to topological studies. Reconstitution of Mistic in the presence of lipids was performed by lowering the detergent concentration to subsolubilizing concentrations via addition of cyclodextrin. To fully exploit the advantages offered by cyclodextrin-mediated detergent removal, a quantitative model was established that describes the supramolecular state of the reconstitution mixture and allows for the prediction of reconstitution trajectories and their cross points with phase boundaries. Automated titrations enabled spectroscopic monitoring of Mistic reconstitutions in real time. On the basis of the established reconstitution protocol, the membrane topology of Mistic was investigated with the aid of fluorescence quenching experiments and oriented circular dichroism spectroscopy. The results of these experiments reveal that Mistic appears to be an exception from the commonly observed transmembrane orientation of α-helical MPs, since it exhibits a highly unusual in-plane topology, which goes in line with recent coarse-grained molecular dynamics simulations.

$Rev: 13581$