Interactions between proteins and lipids lie at the heart of virtually all membrane processes, but on a molecular level they are still poorly understood. Nowadays, simple model systems comprising designed transmembrane peptides in synthetic lipid bilayers are increasingly being recognized as powerful tools to uncover basic principles of protein-lipid interactions. Such model systems enable detailed analysis of how the properties of lipids influence the structure and dynamics of transmembrane helices, how these helices are anchored at the lipid-water interface, and how the length and composition of transmembrane segments influence the organization and dynamics of membrane lipids. In addition, well-characterized model systems have proven useful to refine computational approaches and to develop new techniques for studies of protein-lipid interactions.

Integral membrane proteins have central roles in a vast number of vital cellular processes. A structural feature that most membrane proteins have in common is the presence of one or more R-helices with which they traverse the lipid bilayer. Because of the interaction with the surrounding lipids, the organization of these transmembrane helices will be sensitive to lipid properties like lateral packing, hydrophobic thickness, and headgroup charge. The helices may adapt to the lipids in different ways, which in turn can influence the structure and function of the intact membrane protein. In this review, we will focus on how the lipid environment influences two specific properties of transmembrane segments: their lateral association and their tilt with respect to the bilayer normal.

Theoretical studies predict hydrophobic matching between transmembrane domains of proteins and bilayer lipids to be a physical mechanism by which membranes laterally self-organize. We now experimentally study the direct consequences of mismatching of transmembrane peptides of different length with bilayers of different thicknesses at the molecular level. In both model membranes and simulations we show that cholesterol critically constrains structural adaptations at the peptide-lipid interface under mismatch. These constraints translate into a sorting potential and lead to selective lateral segregation of peptides and lipids according to their hydrophobic length.H ydrophobicity is the key criterion for lipids and proteins to integrate into membranes (1). This self-assembly is driven by the minimization of hydrophobic surface area exposed to the aqueous phase (2). Next to sterols, eukaryotic membranes contain a variety of phospholipids with different chain length (3) and proteins with a variety of transmembrane (TM) domain lengths (4). Why cell membranes contain so many lipids is still enigmatic. But one reason could be membrane sorting due to grouping of TM proteins and lipids with similar hydrophobic length.The "mattress model" predicts that the embedding of a rigid, helical TM protein into a fluid bilayer causes the lipids to adapt locally to a mismatch (5). In this way exposure of hydrophobic surface area is minimized. The adaptive flexing and straightening of the lipids can also be accompanied by a tilting of the protein (6). Because of these strain-causing adaptations of the bilayer, selective association of matching lipids with TM proteins as well as macroscopic sorting processes according to hydrophobic length have been predicted by theory and simulation (7,8). Membrane properties such as elasticity-modulated by cholesterolwere also predicted as crucial parameters (9). Such mismatchdependent, cholesterol-induced sorting has indeed been proposed as a retention mechanism for the Golgi-resident proteins in the secretory pathway (10).Due to the complexity of cell membranes, model membranes have proven a valuable system to investigate hydrophobic matching (11-13). Hydrophobic peptides of the poly-leucine type have been used as generic TM proteins because of the ease of their organic solvent-based reconstitution (14,15). From these experiments, it has become clear that TM proteins indeed tolerate moderate mismatch with the bilayer (13,14,16). However, large mismatch as well as cholesterol have been found to reduce efficiency of TM peptide incorporation into bilayers, suggesting that there are energetic limitations to mismatch buffering (12).Despite indication for selective protein-lipid interactions and hydrophobic matching (17, 18), actual sorting of TM proteins and accompanying lipid cosorting has not been reconstituted in vitro. Therefore, it remains unclear whether hydrophobic mismatch is a significant parameter in the organization of proteinacious membranes.Using TM peptides and lipids of defined acyl chain...

Cell membranes have a nonhomogenous lateral organization. Most information about such nonhomogenous mixing has been obtained from model membrane studies where defined lipid mixtures have been characterized. Various experimental approaches have been used to determine binary and ternary phase diagrams for systems under equilibrium conditions. Such phase diagrams are the most useful tools for understanding the lateral organization in cellular membranes. Here we have used the fluorescence properties of trans-parinaric acid (tPA) for phase diagram determination. The fluorescence intensity, anisotropy, and fluorescence lifetimes of tPA were measured in bilayers composed of one to three lipid components. All of these parameters could be used to determine the presence of liquid-ordered and gel phases in the samples. However, the clearest information about the phase state of the lipid bilayers was obtained from the fluorescence lifetimes of tPA. This is due to the fact that an intermediate-length lifetime was found in samples that contain a liquid-ordered phase and a long lifetime was found in samples that contained a gel phase, whereas tPA in the liquid-disordered phase has a markedly shorter fluorescence lifetime. On the basis of the measured fluorescence parameters, a phase diagram for the 1,2-dioleoyl-sn-glycero-3-phosphocholine/N-palmitoyl sphingomyelin/cholesterol system at 23 °C was prepared with a 5 mol % resolution. We conclude that tPA is a good fluorophore for probing the phase behavior of complex lipid mixtures, especially because multilamellar vesicles can be used. The determined phase diagram shows a clear resemblance to the microscopically determined phase diagram for the same system. However, there are also significant differences that likely are due to tPA's sensitivity to the presence of submicroscopic liquid-ordered and gel phase domains.

Saturated and unsaturated phospholipids (PLs) can segregate into lateral domains. The preference of cholesterol for saturated acyl chains over monounsaturated, and especially polyunsaturated ones, may also affect lateral segregation. Here we have studied how cholesterol influenced the lateral segregation of saturated and unsaturated PLs, for which cholesterol had a varying degree of affinity. The fluorescence lifetime of trans-parinaric acid reported the formation of ordered domains (gel or liquid-ordered (lo)) in bilayers composed of different unsaturated phosphatidylcholines, and dipalmitoyl-phosphatidylcholine or n-palmitoyl-sphingomyelin, in the presence or absence of cholesterol. We observed that cholesterol facilitated lateral segregations and the degree of facilitation correlated with the relative affinity of cholesterol for the different PLs in the bilayers. Differential scanning calorimetry and (2)H nuclear magnetic resonance showed that cholesterol increased the thermostability of both the gel and lo-domains. Increased number of double bonds in the unsaturated PL increased the order in the lo-domains, likely by enriching the ordered domains in saturated lipids and cholesterol. This supported the conclusions from the trans-parinaric acid experiments, and offers insight into how cholesterol facilitated lateral segregation. In conclusion, the relative affinity of cholesterol for different PLs appears to be an important determinant for the formation of ordered domains. Our data suggests that knowledge of the affinity of cholesterol for the different PLs in a bilayer allows prediction of the degree to which the sterol promotes lo-domain formation.

The lateral organization of lipids and proteins in cell membranes is recognized as an important factor in several cellular processes. Cholesterol is thought to function as a modulator of the lateral segregation of lipids into cholesterol-poor and cholesterol-rich domains. We investigated how the affinity of cholesterol for different phospholipids, as seen in cholesterol partitioning between methyl-beta-cyclodextrin and large unilamellar vesicles, was reflected in the lateral organization of lipids in complex bilayers. We especially wanted to determine how the low-T(m) lipid affected the lateral structure. Partition experiments showed that cholesterol had a higher affinity for N-oleoyl-sphingomyelin (OSM) than for palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers, but the highest preference was for N-palmitoyl-sphingomyelin (PSM)-containing bilayers. Partial phase diagrams of POPC/PSM/cholesterol and OSM/PSM/cholesterol bilayers at 23 degrees C and 37 degrees C were used to gain insight into the lateral organization of lipids in bilayers. Analysis of phase diagrams revealed that the phospholipid composition of cholesterol-poor and cholesterol-rich domains reflected the affinity that cholesterol exhibited toward bilayers composed of different lipids. Therefore, the determined affinity of cholesterol for different phospholipid bilayers was useful in predicting the cholesterol-induced lateral segregation of lipids in complex bilayers.

The interaction between cholesterol and phospholipids in bilayer membranes is important for the formation and maintenance of membrane structure and function. However, cholesterol does not interact favorably with all types of phospholipids and, for example, prefers more ordered sphingomyelins (SMs) over phosphatidylcholines (PCs). The reason for this preference is not clear. Here we have studied whether acyl-chain order could be responsible for the preferred sterol interaction with SMs. Acyl-chain order was deduced from diphenylhexatriene anisotropy and from the deuterium order parameter obtained by (2)H-NMR on bilayers made from either 14:0/14:0((d27))-PC, or 14:0((d27))-SM. Sterol/phospholipid interaction was determined from sterol bilayer partitioning. Cholestatrienol (CTL) was used as a fluorescence probe for cholesterol, because its relative membrane partitioning is similar to cholesterol. When CTL was allowed to reach equilibrium partitioning between cyclodextrins and unilamellar vesicles made from either 14:0/14:0-PC or 14:0-SM, the molar-fraction partitioning coefficient (K(x)) was approximately twofold higher for SM bilayers than for PC bilayers. This was even the case when the temperature in the SM samples was raised to achieve equal acyl-chain order, as determined from 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy and the deuterium order parameter. Although the K(x) did increase with acyl-chain order, the higher K(x) for SM bilayers was always evident. At equal acyl-chain order parameter (DPH anisotropy), the K(x) was also higher for 14:0-SM bilayers than for bilayers made from either 14:0/15:0-PC or 15:0-/14:0-PC, suggesting that minor differences in chain length or molecular asymmetry are not responsible for the difference in K(x). We conclude that acyl-chain order affects the bilayer affinity of CTL (and thus cholesterol), but that it is not the cause for the preferred affinity of sterols for SMs over matched PCs. Instead, it is likely that the interfacial properties of SMs influence and stabilize interactions with sterols in bilayer membranes.

The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T m ‫؍‬ 107.4 ؎ 0.3°C) than avidin (T m ‫؍‬ 83.5 ؎ 0.1°C) or bacterial streptavidin (T m ‫؍‬ 75.5°C). AVR4/5 also exhibits a high affinity for biotin (K d Ϸ 3.6 ؋ 10 ؊14 M) comparable to that of avidin and streptavidin (K d Ϸ 10 ؊15 M). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4/5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed.