Structural and functional characterization of the integral membrane proteins CitS and CCR5 by electron microscopy

Secondary transport proteins are integral membrane proteins found in every cell. They facilitate the transport of versatile substrates (e.g. nutrients, ions and drugs) across the hydrophobic membrane barrier. Independent on their mode of transport (symport/antiport) the uphill transport of the main substrate is driven by the coupled flux of a co-substrate downhill its electrochemical gradient. Malfunction of secondary transporter can cause severe physiological disorders like depression and obesity and therefore these transport proteins constitute attractive drug targets. The main part of this PhD thesis is the structural and functional characterization of the secondary citrate/sodium symporter CitS from Klebsiella pneumonia, mainly by transmission electron microscopy (TEM). CitS is the best characterized member of the bacterial 2-hydroxycarboxylate transporter (2-HCT) family. It facilitates the secondary transport of bivalent citrate ions driven by a coupled flux of Na+ across the inner membrane of the host. Hydropathy profiling and extensive biochemical experimentation prior to this study predicted CitS to represent a new structural fold as paradigm for numerous related proteins, so that it constitutes a highly attractive target for structural studies. As a first step, two-dimensional (2D) crystals of recombinant CitS were produced by dialysis assisted reconstitution of pure detergent solubilized protein into bilayer forming phospholipids. Extensive screening of crystallization conditions led to highly ordered tubular 2D crystals suitable for structure determination by cryo-electron crystallography. Therefore, numerous sample preparation methods were evaluated, while plunge-freezing provided significantly better results compared to commonly used sugar embedding methods. As described in chapter 2, image processing of electron micrographs from plunge-frozen 2D crystals provided the projection structure of CitS at 6 Å resolution. The transporter appears as oval shaped dimer measuring 5*9 nm in the membrane plane. ...