Advanced Techniques for the Study of Membrane Proteins in Complex and Native Lipid Environments.

Abstract

The biophysical and biochemical study of membrane proteins is uniquely challenged by the lipid bilayer environment in which membrane proteins reside. While fantastic methods have been developed over the years, there is still a dearth of techniques to study membrane proteins in complex in vitro and in vivo lipid environments. Study of membrane proteins in relevant lipid environment is critical because the lipid environment is known to influence the structure, function, and processing of membrane proteins. The purpose of this dissertation is to characterize and implement novel biochemical and biophysical tools for the study of membrane proteins in biomedically important or native lipid environments. These methods were applied to disease relevant proteins and highlight the true usefulness of novel methods for addressing modern biomedical problems. There is a dearth of isotropic particles capable of solubilizing lipid raft forming lipids such as sphingomyelin and cholesterol. I characterized the first sphingolipid and cholesterol rich bicelles and showcased their suitability for a wide range of biochemical and biophysical methods by studying the structure of the Aβ precursor, C99. Ideally, all in vitro structural and function studies would be validated in native lipid environments but there is a lack of methods to deliver purified membrane proteins to eukaryotic cells. I developed methods to deliver recombinantly-expressed membrane proteins to mammalian cells with amphipathic polymers and model membrane free particles. These methods are unique in that they allow for in vitro labeling of membrane proteins before delivering them to biologically relevant membranes. It is my hope that the broader scientific community will use these methods to study their own membrane proteins and expand upon the methods described here.