Biochemical and Structural Studies of Membrane Proteins

Abstract

Membrane proteins live at the interface between a cell and its environment; hence, they play a variety of important physiological roles such as transmembrane transport, signal transduction, and cell adhesion. The importance of membrane proteins in biology and medicine requires that we understand their structure and function on the atomic level. In this thesis, I studied members of two different membrane protein families, namely the neuronal and keratinocyte TRPV ion channels that sense temperature changes and MP20, a member of the PMP22/EMP/MP20/claudin superfamily. Using a variety of biochemical, X-ray crystallographic and electrophysiological techniques, I addressed mechanistic questions pertaining to the regulation of thermosensitive TRPV channels by ATP and calmodulin in neurons and keratinocytes. For MP20, a protein specific for the lens of the mammalian eye, I used a vesicle assay in combination with electron microscopy (EM) to study its function, ruling out the possibility that MP20 is involved in the formation of membrane junctions. Furthermore, I made progress in expressing and crystallizing MP20 for X-ray diffraction studies. In a separate effort, I also worked on improving and expanding the use of monolayer purification and Affinity Grids, recently introduced techniques to prepare specimens for single-particle EM based on the recruitment of His-tagged proteins to nickel lipidcontaining lipid monolayers. I extended the use of these techniques by synthesizing a glutathione lipid that can be used to recruit GST-tagged proteins. A major hurdle in the use of monolayer purification techniques, however, is the extent of non-specific protein binding to the lipid monolayer. I found that incorporating PEG lipids in the monolayer appears to reduce the problem of non-specific protein binding. While it remains to be seen whether these techniques can be developed to a point at which it will be possible to recruit exclusively tagged proteins out of cell lysates, my goal is to continue to improve and expand the use of the monolayer purification and Affinity Grid techniques in hope to make single-particle EM more easily amenable to biochemists and cell biologists.