9720606 Murphy There are several compelling medical and biotechnological reasons motivating the development of new methods to probe the mechanisms and kinetics of complex systems involving self-associating peptides and lipid bilayers. Key questions include: how many peptides associate together to form a pore? does peptide-peptide association occur prior to, coincident with, or subsequent to peptide-lipid association? must the peptide be in a specific aggregational state in order to interact with the lipid bilayer? This research plan proposes to investigate the feasibility of developing two new methods to probe these kinds of questions. Research objective 1 is to establish whether static light scattering methods can be applied to evaluate the number and association state of peptides embedded in membranes. The pH-sensitive pore-forming peptide GALA and an antibody-GALA derivative, mixed with phosphatidylcholine large unilamellar vesicles, was chosen as the model system for this investigation. Standard static light scattering methods yield information on molecular weight and size of particles in solution, and requires that scattering data be collected on both the sample of interest and the buffer. This approach will be modified by collecting four sets of scattering data: peptide alone, liposomes alone, buffer alone, and peptide-liposome mixture. By subtracting out in turn the scattering from peptide alone, liposomes alone, or buffer alone from the scattering of the peptide-liposome mixture, it may be possible to infer the extent of peptide association in the liposome and the total number of peptides per liposome. Research objective 2 is to investigate the feasibility of using surface plasmon resonance (SPR) spectroscopy to explore the importance of peptide association state in modulating peptide-lipid association. The model system for this investigation will be beta-amyloid peptide monomer and fibrils associating with monolayers of negatively-charged pho spholipids and phospholipid- ganglioside mixtures, SPR is used most frequently to measure binding kinetics of ligands to immobilized receptors. In a modification of this, the immobilized receptors can be replaced by a self-assembled oriented lipid monolayer. A commonly cited difficulty with SPR is that the rates of association can be mass transport, rather than kinetically, limited. The feasibility of turning this difficulty into an asset will be investigated. Since fibrils have significantly slower diffusivities than monomeric peptides, measurement of the mass transport rate should indicate whether the predominant species associating with the lipid layer is monomeric or aggregated. ***
