This project is aimed at the understanding of the physico-chemical mechanisms of membrane remodeling during physiological and pathogenic events. There are three components: ? ? 1. The mechanism of E Coli toxicosis. alpha-Hemolysin is an extracellular protein toxin (117 KDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage?dependent manner. Using a theory for lipidic pore formation, we analyzed this data in order to quantify how HlyA diminished the line tension of the membrane (i.e the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. These results lead to new considerations for existing models for proteo-lipidic pores, and led to our proposing a new model for such pores. ? ? 2. Understanding membrane biophysics. In physics, we usually want to reduce the behavior of the system to fit it to a formula. In biology, all the details are important: organization makes it live. Non-repeating polymers of life code for animation. Thus the organization of proteins in the plasma membrane must be an essential part of the success of life. Consistent with fact that about a third of the dry weight of a cell is membrane, roughly half of all proteins on a eukaryotic genome are membrane proteins. Thus roughly half of biological processes occur on membranes, and each of these processes will have aspects of its function that fall into the realm of physics. It is not surprising that membrane biophysics comprises roughly half of the topics for posters and talks at meetings dedicated to all biophysics ? membranes wraps themselves around almost all of biology. As water is the solvent for soluble proteins, the phospholipid bilayer membrane is the solvent for membrane proteins and the basis of the biological membrane. A semi-crystalline array that is ordered in some aspects and disordered in other aspects, a membrane has both a fluid and a solid character. It is only two molecules thick but can be square millimeters in area (e.g., eggs) or cylinders meters in length (e.g., axons in giraffes). Our closest everyday experience with thin films comes during play with soap bubbles, which are made of thin films of water lined by detergents. Luckily, the phospholipid bilayer itself is stable for many lipid compositions, and bilayers self-assemble upon sufficient hydration of these lipids. Therefore its properties and self-interactions can be extensively studied without proteins in vitro. Our understanding of the physical nature of the membrane backbone comes mostly from studies on the spectroscopy, microscopy, and electrophysiology of bilayers. ? ? 3. How do proteins produce biological shape? Biological membranes exhibit various function-related shapes, and the mechanism by which these shapes are created is largely unclear. We have classified possible curvature-generating mechanisms that are provided by lipids that constitute the membrane bilayer and by proteins that interact with, or are embedded in, the membrane. We describe membrane elastic properties in order to formulate the structural and energetic requirements of proteins and lipids that would enable them to work together to generate the membrane shapes seen during intracellular trafficking.
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