Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Tuesday Speaker Abstracts

Affecting Membrane Assembly and Insertion of a Cholesterol-dependent Cytolysin Listeriolysin O Gregor Anderluh 1,2 , Matic Kisovec 1 , Saša Rezelj 1 , Polona Bedina Zavec 1 , Marjetka Podobnik 1 . 1 National Institute of Chemistry, Ljubljana, Slovenia, 2 University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia. Pore-forming toxins assemble on the surface of cellular lipid membranes in order to generate transmembrane pores. This occurs through several well-defined steps such as recognition and binding to the particular membrane receptor, oligomerisation at the plane of the membrane and insertion of part of the polypeptide chain across the membrane with consequent formation of a pore. Properties of lipid membranes and structural features of pore-forming toxin can affect each of these steps. We will present how lipid membranes composition and single-point mutation affect pore formation of listeriolysin O (LLO). LLO is the most important protein for pathogenicity of a food-borne pathogen Listeria monocytogenes. It belongs to the family of cholesterol-dependent cytolysins which has representatives in many different pathogenic Gram positive bacteria. LLO is unique amongst non Listeria-derived cholesterol-dependent cytolysins in being stable and working optimally at low pH values. LLO allows phagosome-entrapped bacteria to escape to the cytoplasm by creating huge β-barrel transmembrane pores in lipid membranes. Structural features of these pores are not yet completely understood, neither the dependence on lipid composition or pH. Here we will show that pore formation depends on lipid membrane composition, pH and single-point mutations that can have multiple effects on protein stability, rate of pore formation and properties of final pores. The results altogether indicate that the assembly of β-barrel transmembrane LLO pores exhibits significant plasticity, which is an important feature to be included in design and development of medical or nanobiotechnological applications involving cholesterol-dependent cytolysins. Resolving Membrane Protein-Protein Interactions in Live Cells with PIE-FCCS Adam Smith . University of Akron, Akron, USA. Lateral interactions in biological membranes directly influence the activity of cell surface receptors. Resolving the structure and stability of these interactions in situ is difficult because of complexity of the plasma membrane. There are few methods that can resolve the fast dynamics and short length scales that are relevant to membrane organization. I will report on two of my group’s efforts to study membrane structure and organization with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and related methods. PIE-FCCS reports on molecular associations by observing correlated diffusion in and out of a confocal detection area. In this way we can resolve protein mobility and dimerization in live cell membranes with single molecule sensitivity. I will summarize our recent efforts to resolve the clustering and activation mechanism of receptor tyrosine kinases, plexins and G protein-coupled receptors. I will also report on our efforts to study the chemical details of lipid-protein interactions. Together these projects aim to build a more complete picture of the chemical landscape that governs cell communication.

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