Noah Kassem:
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The cell membrane and organellar membranes are instrumental in processes including compartmentalization, protecting cells and transducing signals between compartments. Membrane proteins reside within these membranes and they represent an important class of proteins that have many cellular functions. It is estimated the 22% of the human genome consists of genes encoding membrane proteins. Additionally, 60% of therapeutic drugs target membrane proteins. Unfortunately, due to difficulties at the experimental level, an understanding of membrane protein structures is lagging heavily behind compared to water soluble proteins. This impairs our understanding of the functions and mechanisms of membrane proteins, thus preventing further applied biomedical and biotechnological research. This has led to increasing advances in recombinant production systems, membrane protein carrier systems, and in structural biology methods. Two potential prime drug targets for cancer and cardiovascular diseases are the human sodium-proton exchanger 1 (hNHE1) and the human growth hormone receptor (hGHR). They are widely different, only sharing characteristics as having large intrinsically disordered C-terminal domains. However, only limited structural studies of the full-length hNHE1 and the full-length hGHR exist. Therefore, in the work of this thesis we developed novel protocols to express both hGHR and hNHE1 in an eukaryotic yeast expression system, tested their functionality and purified them to homogeneity in amounts needed for structural characterization. We studied hNHE1 by cryoEM suggesting that hNHE1 forms dimers. We also succeeded in reconstituting hNHE1 in nanodiscs, which laid the foundation for future studies of hNHE1 in nanodiscs by cryoEM. To study the full-length hGHR we reconstituted it into nanodiscs and studied it by combining SAXS, SANS, NMR spectroscopy, and computational modeling in an integrative process to provide a novel full-length structure of the hGHR. This structure exemplifies over 40 cytokine receptors and more than 1300 single pass receptors in humans and indicates that this method can be the bridge needed to enable structures of intact class I cytokine receptors.