By utilizing nanoparticles that mimic the lipid structure of LDL, the mechanism of how C-reactive protein (CRP) distinguishes damaged LDL from intact LDL will be elucidated. The central hypothesis of this research is that high curvature in the lipid coating of LDL results in exposure of the lipid head groups that are then recognized by CRP. Binding to these exposed head groups results in a change to the CRP structure, revealing a C1q binding site that promotes complement activation and a binding site for factor H that limits the inflammatory pathway of complement. A primary and novel tool for this study is a series of lipid-coated nanoparticles that are tuned to match the size and composition of LDL variants, such as small dense LDL and oxidized LDL that are both correlated with increased risk for cardiovascular disease. The utility of a nanoparticle mimic over a liposome is the ability to independently control lipid composition and membrane curvature. The first specific aim is to characterize changes to the secondary and tertiary structure of CRP after it binds to highly lipid membranes with varying degrees of curvature. Specifically, this aim will utilize fluorescence spectroscopy and circular dichroism to test whether CRP responds to all membranes in a similar manner or whether each curvature results in a different conformation of the protein. In the second specific aim, two techniques will explore how the quaternary structure of CRP changes when it encounters a damaged membrane. CRP is composed of 5 identical subunits arranged in a pentamer. The pentameric quaternary structure of the protein is disrupted when the protein binds to damaged membranes such as oxidized LDL, but the mechanism of the dissociation is unclear. Using a small molecule probe, combined with mass spectrometry, it will be possible to assess how the quaternary structure of the protein changes when bound to membranes that have different curvatures and different lipid compositions. A fluorescence energy transfer experiment will similarly probe changes to the quaternary structure and lead to a tool for microscopy of CRP. The third specific aim is to correlate the exposure of C1q and factor H binding sites to interactions with specific membrane structures. This will reveal whether lipid oxidation or changes in lipid membrane shape are more critical to determining whether CRP triggers a pro- or anti- inflammatory response. This project makes an innovative use of nanoparticles to address the very challenging question of how membrane properties, distinct from lipid properties, influence protein binding. This will result in a substantial long-term impac for patients suffering from heart disease.
Each aim will provide new information about CRP that can be utilized in the design of drugs that mitigate the effect of chronic inflammation and the progression of heart disease. Specifically, new conformational states of CRP that are identified and characterized can be considered as targets for the design of drugs that minimize the chronic inflammation associated with higher risk for heart disease.
The approach of using nanoparticles to study protein-membrane interactions is an innovative use of a new technology that will contribute to a fundamental understanding of cardiovascular disease. This work will clarify how C-reactive protein recognizes damaged membranes, which is critical to how the body processes oxidized LDL. This knowledge will lead to improved therapies that mitigate the inflammatory responses to oxidized LDL.
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