Heart disease is leading cause of death in the United States. High-density lipoprotein (HDL) levels are inversely correlated with the prevalence of coronary heart disease. The anti-atherogenic properties of HDL are associated with its role in the pathway of the reverse cholesterol transport, which removes cholesterol from peripheral cells for transport back to hepatocytes. The formation of HDL is facilitated by ATP cassette transporter ABCA1 and apolipoprotein A-I, the major protein of HDL particles. However, the underlying molecular mechanism behind the biogenesis of HDL is not well understood. To provide further understanding of this mechanism, we developed two ABCA1 expression systems in both Sf9 insect cells and FreestyleTM 293-F human cells for functional and structural studies. We designed all constructs of ABCA1 to contain a C-terminal rho1d4 tag that bound to an affinity matrix of rho1d4 antibodies for successful purification. To reconstitute ABCA1 in a detergent-free environment that models the native membrane, we developed three reconstitution systems for ABCA1: saposin A nanodiscs, peptidisc, and amphipol A8-35.
Biochemical and structural studies were carried out to understand the mechanism behind ABCA1’s function. We demonstrated a potential direct interaction of ABCA1 and apolipoprotein A-I with a pull-down experiment. Two cryo-electron microscopy data collections were obtained of ABCA1 in a detergent environment in the presence of ATP with the goal of determining the structure of ABCA1’s active state. We produced a 12 Å reconstruction of ABCA1 from this first data collection. This low-resolution structure confirmed the general structure that currently exists for ABCA1. Processing the data helped us streamline and troubleshoot the electron microscopy workflow pipeline for future data collections. Unfortunately, the second data collection had astigmatism issues that prevented particle alignment during data analysis. However, these data collections provided considerable insight into the ideal sample freezing and grid preparation conditions that affect data collection and data processing.
More transmembrane protein structures are being solved each year but there remain many obstacles and challenges in ABCA1 purification and grid preparation that affect the ability to perform functional studies and high-resolution structure determination. Our developmental work has helped move forward our biochemical understanding of ABCA1 to achieve these aims. The more that is learned about this important membrane protein the more likely it is that future consistent production of ABCA1 will be accomplished to answer the questions of how ABCA1 mediates the formation of HDL particles.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/45518 |
| Date | 25 January 2023 |
| Creators | Urdaneta, Angela |
| Contributors | Atkinson, David |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0024 seconds