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.

<p>Cryogenic transmission electron microscopy (cryoEM) has become an increasingly common tool for determining structures of proteins and protein complex at near atomic resolution. We seek to determine the structure of p97 by cryoEM using an affinity capture approach that employs a family of novel synthetic lipids bearing water soluble PEG units and known high affinity inhibitor molecules at the distal end of the polymer. A library of inhibitor modified affinity lipopolymers of 5000 KD PEG molecular weights were synthesized. The inhibitor modified lipid coated grids were used to capture p97. The reconstruction of p97 revealed the structure at dimeric state at 3.64 Å and monomeric state at 4.33 Å. A PEG unit composed of 20000 KD molecular weight based polyrotaxane containing NTA ligand as affinity tag has been synthesized, used to concentrate 6x-his tagged p97 on TEM which also enabled to see all 3D orientation of the target particles and an initial model of 10.64 Å resolution of p97 structure was resolved. </p>

<div><div><div><p>Calcium-dependent protein kinase II alpha (CaMKIIα) is a highly abundant protein within the hippocampus, the region of the brain responsible for memory and learning. CaMKII has both structural and signaling roles in the regulation of the connective strength of synapses in excitatory neurons. It has a unique structure comprised of twelve subunits that form a dynamic assembly and is highly flexible. Its structural behavior has been shown to affect its activity, and a comprehensive mechanism of structure and function is still not fully understood. The determination of the quaternary structure of the CaMKII holoenzyme has been attempted for nearly 20 years by a variety of methods, with no one method giving a definitive structure. Problems in obtaining a structure originated with observation methods that estimated quaternary shape from low-resolution ensemble averages or required significant alteration of the protein to enforce a particular conformation. In this work, experiments were conducted to remove these limitations and provide a path towards the quaternary structure of CaMKIIα. Different expression and purification methods were evaluated to produce an optimal protocol for the generation of samples of concentrated, monodisperse, autoinhibited full-length wild-type CaMKIIα for study with cryo-electron microscopy. Strategies for microscopy sample preparation were investigated, including affinity girds, graphene-coated grids, and holey carbon grids. Lastly, experiments using negative stain electron microscopy, cryo-electron microscopy with single particle analysis, and cryo-electron tomography with subtomogram averaging were conducted to reveal the conditions required to produce an unambiguous three-dimensional structure. It was found that the assembly of the hexameric hub rings appeared to have flexible orientation, and superposition problems inherent in two-dimensional projection averaging requires the use of cryo-electron tomography to unravel the ambiguity in both hub orientation and catalytic module placement within the reconstructed volume. A subtomogram average of a limited number of particles revealed a hub domain that matched the morphology of prior reports, but the determination of catalytic module placement was not resolved. The cumulative result of this work establishes a strategy for the large-scale data collection needed to fully elucidate the structure of this challenging and fascinating protein.</p></div></div></div>