The cell is the basic unit of life. To maintain its high internal order in an open continuous system an essential need of all living organisms is an effective barrier to the unordered environment. This barrier enables the cell to accumulate nutrients gathered from its environment and to retain the products it synthesizes for its own use, while excreting metabolic waste products. Higher organized eucaryotic cells have further intracellular compartments, used for specific metabolic functions that are separated from the cytosol.
In all three domains of life (eubacteria, archaea and eucarya) the core structure of these barriers is formed by lipids, mainly phosphoglycerides. Due to their amphiphilic nature in an aqueous environment lipids selfassemble into bilayers (membranes). The hydrophobic hydrocarbon moieties are protected in the interior of the membrane and the hydrophilic head groups are in contact with the surrounding water. Due to an appropriate lipid composition the bilayers are in a liquid crystalline state over a wide temperature range.
The single lipid molecules are ordered perpendicularly to the membrane plane where they are free to lateral diffusion. Flipping of lipids from one leaflet to the opposite one is a rather rare event. This allows cells to form and retain asymmetric bilayers with different lipid composition in both monolayers and adapt their membranes to specific requirements.
Membranes are∼5 nm thick with the central hydrophobic core comprising∼3 nm which is sufficient to block the passage of hydrophilic solutes like ions, sugars or amino acids through the cell envelope whereas hydrophobic compounds like CO2, N2, O2 or NH3easily can diffuse through the membrane.
Membranes are, however, more than just passive barriers. Most of the biological func-tions taking place at biological membranes, e.g. the acquisition of nutrients, the conversion of chemical energy in an electrochemical membrane potential and vice versa, or the dis-posal of metabolic waste products require specialized transmembrane spanning transport proteins. If not anchored to other structures of the cell, e.g. the cytoskeleton or com-ponents of the cell wall, they diffuse within the membrane plane. Membrane embedded
Figure 1.1: Liquid mosaic model of an eucaryotic cell membrane, taken from the following web resource:
http://en.wikibooks.org/wiki/A-level Biology/Biology Foundation/cell membranes and transport.
proteins that do not span the whole membrane further contribute to functions that are associated to biological membranes. These experimental findings led to the fluid mo-saic model of biological membranes (figure 1.1) as described first by Singer and Nicolson (1972). The importance of membrane proteins for appropriate cell function is reflected by the high protein content which is in the range between 20 and 70 % of the total membrane mass and strongly depends on the membrane function.
In addition to the ubiquitous cytoplasmic membrane, Gram− bacteria possess an outer membrane which encloses the periplasmic space. A highly crosslinked macromolecule forms the so-called peptidoglycan layer in the periplasm and provides these bacteria with their rigidity. Two major differences distinguish the outer from the inner membrane.
First, in the outer membrane some lipids of the outer leaflet are replaced by lipopolysac-charides, molecules that are only synthesized by Gram− bacteria. The densely packed polysaccharides on the bacterial outside provide an additional barrier against the per-meation of hydrophobic compounds, and is one reason why Gram− bacteria are rather resistant against many antibiotics since these cannot enter their target cells. Second, in-tegral outer membrane proteins, so-called porins, form large water filled channels which allow the free diffusion of water and smalls solutes (<600 Dalton), e.g. sugars, aminoacids, salts, between the periplasm and the environment. However, the outer membrane still provides a barrier for larger polymers and proteins.
The group of membrane proteins is as diverse as the functions they need to perform.
Accordingly, whole genome analyses from all three domains of life (eubacteria, archaea,
eucarya) gave estimates that∼30 % of all open reading frames are encoding transmem-brane proteins (Elofsson and von Heijne, 2007; Liu et al., 2002; Wallin and von Heijne, 1998) and one third of these catalyse the transport of molecules from one side of the membrane to the other (Paulsen et al., 1998a,b). Due to this background, it is not surprising that many membrane transport proteins are related to diseases as well as — due to their exposed location at the cell surface — provide important virulence factors during bacterial infections. Currently more than 50 % of all drugs for application in humans are targeted against transmembrane proteins (mainly G-protein coupled recep-tors or ion channels) (Russell and Eggleston, 2000; Klabunde and Hessler, 2002; Krogh et al., 2001). Thus a profound knowledge of membrane protein structures and func-tions would not only contribute to our understanding of relevant cell surface processes but also open a new field for drug development and design. Despite the obvious needs for high resolution structures and the large efforts that are put into this field, our cur-rent knowledge on membrane protein structures lags far behind that of soluble proteins.
Less than 1 % of all protein structures deposited in the “Protein Data Bank” (PDB) (Berman et al., 2000) are of membrane proteins and currently these are not more than 201 unique structures as listed on the web page of “Membrane Proteins of known Struc-ture” (http://blanco.biomol.uci.edu/Membrane Proteins xtal.html). In cases where the transport process is not performed by a single protein component but rather by a large complex composed of several to many different polypeptide chains as in the case of some protein translocating systems, the situation is even worse. However, the contribution of these structures to our understanding of membrane transport processes is immense.
Until now the only reliable method to obtain high resolution structures of membrane proteins or protein/protein complexes is to crystallize the purified protein/complex and to calculate a structural model from the diffraction pattern obtained by X-ray crystal-lography. The purification and crystallization of membrane proteins or protein/protein complexes still belongs to the major challenges in molecular biology and is the main rea-son for the deficit in structural knowledge of membrane transport systems (Carpenter et al., 2008; Walian et al., 2004; Werten et al., 2002). The large quantities of protein needed for crystallization usually require an efficient expression system for recombinant protein. In the case of transmembrane proteins or heteromeric protein/protein com-plexes already this first step is often successful only after extensive trial and error or does not succeed at all (Junge et al., 2008; Midgett and Madden, 2007; Surade et al., 2006).
Further bottlenecks towards the structure determination can arise either during the sol-ubilization of membrane proteins from their lipidic environment in intact conformation
Figure 1.2: Schematic overview about the four classes of small solute transport proteins in biological mem-branes. The gradients of the solutes across the membrane are indicated by triangles with the tip pointing towards the lower electrochemical potential. Channels and uniporters permit the import or export of solutes down their electrochemical gradient. Symporters and antiporters catalyze the movement of one solute against its gradient (red symbols), driven by the movement of one or several atoms/molecules of another solute down its electrochemical gradient (grey symbols). In most cases, the driving force is deliv-ered by the inward flux of protons, sodium or other cations. Primary active transporters utilize a primary source of energy, here exemplified by ATP hydrolysis to pump solutes against their electrochemical po-tential either inward or outward cells or cellular compartments. Finally, group translocators chemically modify their substrates at the cytosolic side of the cytoplasmic membrane during uptake, exemplified here by the phosphotransferase system which transfers the phosphoryl group of phosphoenolpyruvate to the transported sugars. Thus, the transport does not contribute to the concentration gradient of the unmodified substrate across the membrane and the gradient is omitted in the illustration.
(Seddon et al., 2004; Lundstrom, 2006) or the purification of homogeneous and struc-turally stable proteins/complexes and last but not least during the crystallization in the presence of detergents (Caffrey, 2003; Lacap`ereet al., 2007; Ostermeier and Michel, 1997).