New Strategies in the Expression, Purification, and Reconstitution of the Cystic Fibrosis Transmembrane conductance Regulator for Biochemical and Biophysical Characterization in Different Lipid Environments
Strickland, Kerry M.
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Membrane proteins are difficult to study in solution due to the use of detergent to protect the hydrophobic portion of the protein that interacts with the fatty acid tails of the lipid bilayer. However, a recent technology called nanodiscs provides a packaged lipid bilayer system in which membrane proteins can be studied in solution in a lipid environment and thus without the use of detergent. Nanodiscs have revolutionized the study of membrane proteins, even difficult and complex membrane proteins. A particularly challenging membrane protein, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), has shown to be very sensitive to the detergent and lipid environment, especially during purification and reconstitution. This dissertation explored new methods of expression, purification, reconstitution, functional assays, and structurally important lipids for human CFTR. The development of multiple expression systems improved detergent purification methods and increased the yield of functional CFTR. The utilization of nanodiscs technology has provided a method of measuring changes in CFTR’s ATPase activity dependent on the environment and may provide a new method for determining structure-induced changes to CFTR that are dependent on the environment. The identification of possibly key structural lipids in detergent-solubilized CFTR and some preliminary investigation into the annular lipid environment of CFTR was completed by lipidomics mass spectrometry. This dissertation also utilized both traditional and nontraditional molecular modeling methods to refine a CFTR homology model and study a key interaction in the translation of ATP binding in the nucleotide binding domains (NBDs) to the transmembrane domains (TMDs).