alkaline pH acidic pH
cytoplasmic extracellular H+
H+
Figure 2.10.: pH-dependent proton pumping vectoriality of PR. For alkaline pH, PR pumps protons like BR from the cytoplasmic side to the extracellular side. For acidic pH, an inverted proton transfer direction is observed from the extracellular side to the cytoplasmic side for PR.
2.4. Properties of lipid membranes
Biological membranes play an important role for a huge range of biological processes in the cell such as transport and signaling. They act as selective permeable barriers and lead to compartmentalization of the cell. A thorough understanding of the molecular organization of these membranes is crucial for a better understanding of these important biological processes.
2.4.1. Structure of lipids
Lipids are the building blocks of a lipid bilayer which is the most fundamental structure of cell membranes [40]. The phospholipids are a major component of all cell membranes.
They possess an amphiphilic structure having a polar phosphate headgroup and usually two fatty acid tails. Headgroup and fatty acid tails are linked together by a glycerol molecule. The charge of the lipid headgroups can vary as well as the number of carbon atoms for the fatty acid tails. When all the bonds between the carbon atoms in the tails are single bonds, the lipid is classified as "saturated". Lipids which contain one or more double bond(s) between the carbon atoms of the hydrophobic chain are termed
"unsaturated" or("polyunsaturated"). Figure 2.11 displays the structure of 1,2-dioleoyl-sn-glycero-3-phosphocholine abbreviated as 18:1 (∆ 9-cis) or DOPC. The first abbreviation
2. Current state of research
is the easiest way of classifying lipids. Thereby, the first number before the colon indicates the number of carbon atoms in the hydrophobic chain, while the second number after the colon indicates the number of double bonds found in the chain [41]. DOPC has 18 carbon atoms in the fatty acid chain and onecis double bond at carbon position 9 counted from the first carbon of the C=O ester group which lies at the interface between headgroup and lipid tail. The position of the double bond is therefore denoted by the ∆ nomenclature.
Apart from phospholipids, sterols such as cholesterol play an important role in the fatty acid composition of cell membranes. Cholesterol is a rigid molecule having a steroid ring structure and a polar head. It plays an outstanding role in the maintenance and regulation of membrane structural integrity and fluidity of animal plasma membranes.
N
Figure 2.11.: Structure of 1,2-dioleoyl-sn-glycero-3-phosphocholine abbreviated as 18:1 (∆
9-cis) or DOPC. The polar phosphate headgroup is highlighted in blue and the two hydrophobic lipid tails in gray, both containing one cis double bond at carbon position 9.
2.4.2. Lipid phase transitions
Soft matter like lipid bilayers undergoes phase transitions just like basically any other kind of matter [41]. As a very simple example, water in form of ice melts to liquid water and finally boils to water vapour with increasing temperature. Likewise lipid bilayers can be in a more ordered gel phase and undergo a phase transition to a more fluid liquid crystalline phase with increasing temperature. The temperature at which the transition occurs is defined as the phase transition temperature Tm. Phosphatidylcholines (PC) are the most abundant class of lipids in mammalian membranes. Figure 2.12 schematically shows the phase transitions of this lipid class with increasing temperature.
Due to the large area requirement of the bulky polar headgroup of phosphatidylcholines [43], the fatty acid chains are tilted 30◦ with respect to the bilayer normal in the gel
2.4. Properties of lipid membranes
Gel phase Ripple phase Liquid crystalline phase
Temperature
Figure 2.12.: Lipid phases of phosphatidylcholines, adapted from [42]. With increasing temperature, the lipids undergo a phase transition from the ordered gel phase to the more disordered liquid crystalline phase via a ripple phase. The bilayer thickness substantially decreases in the liquid crystalline phase, due to the higher disorder of the hydrocarbon chains.
phase and show a pretransition to the ripple phase. With further increasing temperature, the main transition to the liquid crystalline phase can then be observed, in which the fatty acid chains are no longer tilted, since the bulky headgroups are further apart from each other [44]. The lipid fatty acid chains convert from a rigid extended all-trans conformation in the gel phase to the more flexible disordered liquid crystalline phase characterized by the presence of gauche conformations [45].These gauche bonds result in a kink of the hydrocarbon chain being responsible for the higher disorder in the liquid crystalline phase (Figure 2.13). Hence, the hydrophobic bilayer thickness is reduced in the liquid crystalline phase. The phase transition temperature is determined by a competition of the entropically favoredgauche conformation of the hydrocarbon chains and the attractiveVan der Waals interactions between neighboring chains [44]. The fatty acid chain length is therefore proportional to the phase transition temperature, since longer chain lengths result in increased van der Waals interactions leading to a higher phase transition temperature. The introduction of a cis double bond drastically reduces the phase transition temperature having a maximal effect when the cis double bond is located in the middle of the chain [44]. Thecis double bond induces a kink in the fatty acid chain which hinders an efficient molecular packing required for the gel phase.
Infrared spectroscopy is one of the powerful tools to monitor the phase transition of lipids.
Thereby, thetrans-gaucheisomerization from the gel phase to the liquid crystalline phase can be monitored in form of a frequency shift of the CH2 stretching vibrations of the hydrocarbon chains [46, 47].
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Figure 2.13.: Upper panel: Potential energy for rotation around a carbon-carbon bond.
Trans-conformation t is the lowest energy state with a dihedral angle of 180◦. Further local minima aregauche-conformations g+ and g- with dihedral angles of 60◦ and 300◦. Middle panel: Newmanprojections of the respective confor-mations. Lower panel: Examples of lipid chain configurations (all-trans ttt, first-order kink g+tg- and monounsaturated lipid tail with double cis-bond).
Adapted from [44].
2.4. Properties of lipid membranes
2.4.3. Artificial model membranes
The lipid environment surrounding a membrane protein can interact with the protein and has a high impact on its function. Investigation of this influence requires profound knowledge about the physical properties of the respective lipids surrounding the protein.
In vivo, membranes are typically built up by many different lipids with distinct physical properties, making it tremendously difficult to analyze the effect of the membrane on the function of the protein. Therefore, in vitro experiments usually try to reduce the complexity by using membrane mimetic systems with only one sort of lipid or a few lipids with precise physical properties in order to obtain a better understanding how these properties can affect the functionality of membrane proteins. Highly applied membrane mimetic systems are micelles or liposomes (Figure 2.14). More recently lipid nanodiscs have also become a membrane mimetic tool [48]. Membrane scaffold proteins (MSP) are used in this case to stabilize the disk-shaped lipid bilayers. By varying the length of the MSP, precise control of the oligomerization state of the membrane protein of interest can be achieved.
Liposome Micelles
Hydrophilic head
Hydrophobic tails
Figure 2.14.: Membrane mimetic systems. Liposomes are spherical vesicles built up by a lipid bilayer enclosing an aqueous environment. Micelles are closed lipid monolayers which start to assemble at the critical micelle concentration (CMC). Both systems can assemble spontaneously. These systems are largely used to imitate a specific lipid environment with precise lipid physical properties for membrane proteins.
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