Protein with phospholipid monolayers

Pitcher WH III, Keller SL, Huestis WH. Interaction of nominally soluble proteins with phospholipid monolayers at the air-water interface. Biochim Bio-phys Acta 2002 1564 107. 

These results show that the ability of these signal peptides to interact with phospholipid monolayers indeed correlates with their in vivo activity. The pressure increases due to the functional signal peptides (8—11 dyn/cm) are in the same range as those caused by proteins known to insert into monolayers (Bougis et al., 1981). In contrast, prothrombin, which binds only to the membrane surface, causes a pressure increase in a phospholipid monolayer of 1.9-2.3 dyn/cm (Mayer et al., 1983). These values are almost identical to those obtained for perturbation of the monolayer by the deletion-mutant signal peptide. 

By using a surface radioactivity technique, the penetration of the hydrophobic and flexible 1-14C-acetyl--casein and the rigid and globular 1-14C-acetyl-lysozyme molecules into phospholipid monolayers in different physical states was monitored. The adsorption of ff-casein to lecithin mono-layers is described by a model in which it is assumed that the protein condenses the lecithin molecules so that the degree of penetration is a function of the lateral compressibility of the phospholipid monolayer. The interaction of ff-casein with phospholipid monolayers is dominated by the hydrophobicity of the macromolecule, but lysozyme tends to accumulate mostly beneath phospholipid monolayers in this situation, electrostatic interactions between the lipid and protein are important. 

TiTuch of our understanding of the phase behavior of insoluble - monolayers of lipids at the air-water interface is derived from Adam s studies of fatty acid monolayers (I). It is now clear that the phase behavior of phospholipid monolayers (2) parallels that of the fatty acids we make use of these structure variations in our study of the interactions of phosphatidylcholine (lecithin) monolayers with proteins. Because of the biological significance of the interfacial behavior of lipids and proteins, there is a long history of studies on such systems. When Adam was studying lipid monolayers, other noted contemporary surface chemists were studying protein monolayers (3) and the interactions of proteins with lipid monolayers (4). The latter interaction has been studied by many so-called 4 penetration experiments where the protein is injected into the substrate below insoluble lipid monolayers that are spread on the... 

The studies on the mode of interaction of prothrombin with phospholipid monolayers, using complementary methods of surface measurement are reviewed. They were investigated at air-water and Hg-water interfaces respectively by radioactivity and electrochemistry. A process more complex than a simple adsorption could be detected. Indeed, the variation of the differential capacity of a mercury electrode in direct contact with phospholipid monolayer, induced by the interaction with prothrombin could be interpreted as a model of its penetration into the layer this was confirmed by the study of the dynamic properties of the direct adsorption of this protein at the e-lectrode, followed in part by the reduction of S-S bridges at the electrode. It could be also concluded that prothrombin resists complete unfolding at these interfaces. 

As seen in Figure 2, the ac polarogram resulting from the interaction of the monolayer with prothrombin shows that an electroactive group contributes to the capacitance curve a pseudocapacitance peak, at around - 0.7 V, which is ascribed to the oxy-reduction of the disulfide bridges at pH 7.8. Since the formation of cysteine requires hydrogen ions, the half-wave potential and thus also the pseudocapacitance peak is shifted with decreasing pH to more positive polarization. Since two contributions, one from the protein and the other from the phospholipid monolayer, are involved in the capacitance values, it was of importance to study particularly the behavior of the protein in direct contact with the electrode, in order to be able to interpret better the data obtained when prothrombin interacts with phospholipid monolayers. 

Giehl, A, Lemm, T, Bartelsen, O, Sandhoff, K and Blume, A (1999) Interaction of the GM2-activator protein with phospholipid-ganglioside hilayer membranes and with monolayers at the air-water interface. Em J Biochem, 261, 650-658. 

Freer, J. H., Birbeck, T. H., and Bhakoo, M. (1984) Interaction of staphylococcal 5-lysin with phospholipid monolayers and bilayers a short course. In Bacterial Protein Toxins (J. H. Freer, ed.), Academic Press, London, pp. 181-189. 

There are at least nine apoproteins associated with the lipoproteins, as well as several enzymes and a cholesteryl ester transfer protein. There are two major types of apoproteins. Two apoproteins (apo B100 and apo B48) are tightly integrated into the phospholipid monolayer. The other seven proteins are less tightly associated with the phospholipid and exchange among the lipoproteins. The apoproteins have three major functions. (1) They are important structural components. (2) Some of the apoproteins modulate the ac-... 

In vivo, the folding process may be supported by a periplasmic chaperone called Skp. Skp is a 17 kDa protein associated with the plasma membrane that, together with peptidyl prolyl isomerases and disulfideexchanging enzymes, helps folding freshly synthesized proteins in the periplasm (Schafer et al., 1999). Skp binds to partially unfolded polypeptides. Depending on the presence of phospholipids, lipopolysaccharides, and bivalent cations, Skp exists in two conformations, one of which is protease-sensitive (DeCock et al., 1999). Moreover, it was shown that Skp binds to unfolded periplasmic proteins and inserts into phospholipid monolayers, corroborating its putative role as helper in folding and membrane insertion. 

IR spectroscopy of the X-receptor protein signal peptide in phospholipid monolayers shows that the peptide affects the packing of the lipid hydrocarbon tails (M. S. Briggs, R. A. Dluhy, D. G. Cornell, and L. M. Gierasch, unpublished results). In samples formed at the same surface pressure, the lipid tails are oriented differendy in the presence and absence of signal peptide. A phospholipase assay for structural defects in phospholipid bilayers (Jain et al., 1984) indicates that the X-receptor protein signal peptide interacts with vesicles to induce such defects. The peptides perturb the lipid structure at lower mole fractions than do various lysophospholipids. These data provide yet another indication that signal peptides interact with and perturb lipid complexes. 

A phospholipid monolayer in the surface is consistent with the current model that LD are formed by TAG deposition between the two leaflets of the ER membrane and may remain connected to it [144, 145 see below]. Distribution of acyl-CoA cholesterol acyltransferase-1, a major enzyme that synthesizes cholesterylester, in the entire ER [148] seems to indicate that LD may bud anywhere along the membrane. However, Cap-LC/ESI mass spectrometry showed that FA moieties of phosphatidylcholine and lyso-phosphatidylchohne in LD are distinct from those in the rough ER [149]. The results do rule out the generation of the LD surface generated from the ER membrane but indicate that the former is a highly differentiated domain. Mature LD might be independent of the ER. Alternatively, the LD may be connected to the ER, but some molecular mechanism may demarcate the LD surface from the bulk ER membrane as postulated for other ER domains [150]. Whatever is true, TAG synthesized in wide areas of the ER do not deposit indiscriminately but are concentrated to loci specialized to make LD. ADRP or other LD-associated proteins may be involved (see below). 

A FIGURE 18-12 Model of low-density lipoprotein (LDL). This class and the other classes of lipoproteins have the same general structure an amphipathic shell, composed of a phospholipid monolayer (not bilayer), cholesterol, and protein, and a hydrophobic core, composed mostly of cholesteryl esters or triglycerides or both but with minor amounts of other neutral lipids (e.g., some vitamins). This model of LDL is based on electron microscopy and other low-resolution biophysical methods. LDL is unique in that it contains only a single molecule of one type of apolipoprotein (apoB), which appears to wrap around the outside of the particle as a band of protein. The other lipoproteins contain multiple apolipoprotein molecules, often of different types. [Adapted from M. Krieger, 1995, in E. Haber, ed., Molecular Cardiovascular Medicine, Scientific American Medicine, pp. 31-47]... 

Fig. 4. Crystal structure of the outer membrane phospholipase A dimer from E. coli shown in the plane of the membrane. The top half of the molecule is located in the lipopolysaccharide monolayer facing the exterior of the cell. The phospholipid monolayer of the outer cell membrane would be located around the bottom half of the protein. Two calcium ions are shown at the active sites while Ser-144 of each active site is covalently modified with a hexadecylsulfonyl moiety represented in a ball and stick format. Structure is adapted from Ref. [12]. (See color plate section, plate no. 8.)...

As an example of a membrane model, phospholipid monolayers with negative charge of different density were used. It had already been found ( ) and discussed O) that the physical and biological behavior of phospholipid monolayers at air-water interfaces and of suspensions of liposomes are comparable if the monolayer is in a condensed state. Two complementary methods of surface measurements (using radioactivity and electrochemical measurements), were used to investigate the adsorption and the dynamic properties of the adsorbed prothrombin on the phospholipid monolayers. Two different interfaces, air-water and mercury-water, were examined. In this review, the behavior of prothrombin at these interfaces, in the presence of phospholipid monolayers, is presented as compared with its behavior in the absence of phospholipids. An excess of lipid of different compositions of phos-phatidylserine (PS) and phosphatidylcholine (PC) was spread over an aqueous phase so as to form a condensed monolayer, then the proteins were inject underneath the monolayer in the presence or in the absence of Ca. The adsorption occurs in situ and under static conditions. The excess of lipid ensured a fully compressed monolayer in equilibrium with the collapsed excess lipid layers. The contribution of this excess of lipid to protein adsorption was negligible and there was no effect at all on the electrode measurements. 

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