Developing Microfluidic Devices for the Optimization of Nanodiscs to Measure Membrane Protein Structure and Function

Abstract

Membrane proteins are essential to cellular functions, including cell-cell communication and signal transduction. However, the study of membrane proteins has lagged behind that of soluble proteins as once removed from the native cell lipid bilayer membrane proteins tend to misfold and aggregate, leading to loss of structure and function. To enable the study of stable and active membrane proteins, a variety of membrane mimetics have been developed. The membrane protein mimetic used throughout this dissertation will be Nanodiscs, which are introduced in chapter one. The second chapter is a review of microfluidic devices that have been developed to study membrane proteins. The third chapter describes the microfluidic device we developed to form Nanodiscs containing membrane proteins on a faster timescale (5 minutes) and utilizing less material (90 μL). We formed both empty Nanodiscs and Nanodiscs containing the membrane protein cytochrome P450 CYP3A4 using our device and confirmed that the CYP3A4 remained functional after microfluidic incorporation, as measured with ligand binding assays. The fourth chapter discusses using the microfluidic device to form Library Nanodiscs, a type of Nanodisc that is formed with cell lysate or isolated cell membranes instead of with a recombinantly expressed and purified membrane protein. I was able to confirm the incorporation of full-length Epidermal Growth Factor Receptor (EGFR) into Nanodiscs and had some success in measuring EGFR activity changes in response to tyrosine kinase inhibitors. These results that indicated EGFR in Nanodiscs had activity responses that are not noticed in lysate, potentially suggesting that Nanodisc incorporation allows for improved activity. However, high background in the activity measurements from other membrane proteins present in the Nanodisc sample has led to the necessity to purify for only EGFR-containing Nanodiscs, which is still a work in progress to obtain high concentrations of these pure samples for activity measurements. The fifth chapter describes a collaboration with the lab of Professor Neil Marsh, University of Michigan, focused on incorporating viperin into Nanodiscs. Viperin is a membrane-associated protein that interacts with different membranes within the cell, and we were interested in probing how this interaction impacts viperin’s structure and function. We have determined the lipid composition that allows for optimal viperin incorporation which is important for our downstream activity assay and structural measurements. The sixth chapter was performed in collaboration with the labs of Professor Brandon Ruotolo and Professor Philip Andrews, University of Michigan, in which we applied mass spectrometry to Nanodiscs with a focus on the impact of lipid composition on membrane proteins. With the Ruotolo lab, we incorporated cytochrome P450s into Nanodiscs and determined structural information about CYP3A4 in different lipid compositions using ion mobility-mass spectrometry. We found that changing the lipid environment caused changes in the protein gas-phase unfolding which indicates differences in the CYP3A4 structure in each lipid environment. With the Andrews lab, we formed Library Nanodiscs of different lipid compositions using mitochondrial lysate and used peptide-fingerprinting mass spectrometry to test which membrane proteins incorporate into Nanodiscs of each lipid composition. Though replicates still need to be performed, some proteins seem to incorporate into Nanodiscs of any lipid composition while others only incorporate into one Nanodisc. Finally, I end with conclusions and future work in chapter 7. There are a variety of other areas in which this project will continue within the Bailey lab, including microfluidic and biological improvements.