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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.