The research outlined in this proposal investigates the pathophysiology of hematologic and vascular disorders that arise when serum immunoproteins bind to the plasma membranes of blood cells. Biophysical techniques will be used to identify molecular events on the membrane surface that give rise to sublytic changes in membrane permeability, and which may account for disordered cellular function frequently observed in autoimmune diseases of erythrocytes, platelets, and granulocytes. To undertake this study, serum complement proteins C8 and C9 will be labeled with visible-wavelength fluorescent chromophores, serving as spectroscopic """"""""reporters"""""""" of the associative and conformational states of these proteins when bound to erythrocyte membranes and phospholipid vesicles of defined composition and size. Fluorescence resonance energy transfer (RET) between fluorophores attached to C9 will be used to probe the polymerization of that protein upon membrane binding. Similarly, aggregation of multiple C5b-8 will be probed using RET donor and acceptor pairs of fluorescently-labeled C8. Associative states of these proteins in situ will be directly correlated to the functional properties of the resulting membrane pore, with the goal of identifying conditions of C5b-9 assembly that lead to sublytic alteration of membrane permeability. Emphasis will be placed on identifying compositional and physical properties of the target membrane that influence the conformational state of the bound proteins, in order to determine the molecular basis for alterd membrane susceptibility to their cytolytic activity. As part of this project, fluorescence techniques developed to probe the interaction between C9 and membrane-bound C5b-8 will be used to identify the membrane defect of the PNH-III red cell produced in the hemolytic disorder Paroxysmal Nocturnal Hemoglobinuria. Future application of the methods developed in this project to flow cytometric analysis of changes in platelet and granulocyte membrane function upon C5b-9 binding and membrane insertion is planned.
Showing the most recent 10 out of 40 publications
