Bilayer-Insertion Mechanisms of Self-Inserting Membrane Proteins by Combined Ensemble and Single-Molecule Spectroscopy

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This joint proposal addresses the fundamental question of how particular membrane proteins self-insert into lipid bilayers without requiring translocons or external energy sources. To this end, we intend to combine a comprehensive set of ensemble optical-spectroscopic methods with single-molecule Förster resonance energy transfer (smFRET) experiments to study the structural dynamics of both folded and unfolded states as well as the membrane-insertion mechanisms of two self-inserting proteins. The first protein is Mistic, a helical-bundle protein essential for bacterial biofilm formation. Although Mistic is being exploited to aid in the production and membrane insertion of other membrane proteins, its self-insertion capability remains elusive. We will explore the effects of membrane properties such as hydrophobic thickness and dipole potential, which have been hypothesized to modulate the fold and stability of Mistic, and unveil the environment-dependent dynamics of its folded state and its mechanism of membrane insertion. We thus expect to arrive at a better understanding of both its presumed physiological function as a membrane sensor for triggering biofilm formation as well as its biotechnological use as a membrane-insertion chaperone. The second protein of interest is outer membrane phospholipase A (OmpLA), a bacterial beta-barrel that has proven highly useful in folding studies on integral membrane proteins. In light of the importance of this protein as a model system for in vitro investigations, we aim at a better understanding of its unfolded state under denaturing conditions and its (coupled?) folding and membrane insertion, with particular emphasis on the role of prestructuring or conformational selection. The combination of these two structurally and functionally distinct membrane proteins is motivated by methodological synergism and complementarity. This joint project crucially depends on close interactions between the groups of the two applicants, since it comprises a wide range of diverse but complementary methods and approaches, including protein production, purification, and site-specific double labeling for smFRET, automated circular dichroism and fluorescence quenching spectroscopy at the ensemble level, single-molecule experiments using a custom-built instrument, and the development and validation of computational analysis methods.