Interaction of proteins with phospholipid

The amount of investigational work on the subject of formulation also needs further review and amplification. Basic formulation studies have suggested that proteins could be used to form drug delivery systems (e.g., microparticles) the interactions of proteins with phospholipids is of sufficient interest to justify a short chapter. 

Interactions of proteins with membranes are quite unspecific. Accordingly, typical PH-domain proteins, such as pleckstrin and spectrin, bind with no apparent stereospecificity and with quite low affinity to phosphotidylinositol bisphosphate (PtdInsP2) and IP3 in lipid vesicles (K > 30 (J.M and 40 (xM, respectively). Moreover, whether all PH-domain proteins associate with negatively charged membrane phospholipids is not certain. However, there is the case of PLC-81, where the PH domain is instrumental in bringing the soluble, cytosolic enzyme to its membrane-bound substrate, PtdInsP2. This function is supported by deletion mutants of PLC, lacking the PH domain, which caimot find the membrane-bound substrate. Therefore, while the role of the PH-domain motif in protein-protein interactions and interconnectivity is questionable, its role in membrane attachment of soluble, cytosolic proteins is quite persuasive. 

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 functional microarray typically consists of a collection of full-length functional proteins or protein domains printed on glass slides that are then exposed to a protein preparation from a cell that represents the entire proteome of that cell. This method is useful in determining protein-protein interactions. In addition, this method is useful in predicting the interaction of proteins with DNA, RNA, phospholipids, and small molecules. RPA includes glass slides on which a cellular protein preparation is fixed and then probed with a known antibody. This method helps in identifying the proteins that are altered and cannot bind with a known antibody in the proteome of diseased cell types. This method also identifies the proteins that are altered as a result of phosphorylation or other posttranslational modifications in normal and disease conditions or under growth conditions. 

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. 

The situation is even worse for membrane lipids. Not a single, naturally occurring phospholipid with unsaturated hydrocarbon chains has yet been crystallized. However, nearly 40 crystal structures of closely related synthetic glycerolipids with saturated hydrocarbon chains have been solved by X-ray. On the structural level, little is known about the interactions of proteins with lipid bilayer environments. Detergent molecules have been detected in some of the X-ray structures, and a small number of studies discuss lipids bound to proteins. An example is cytochrome C oxidase crystals, where the lipids were found to be arranged in a bilayer structure. 

DCIA has been used to label numerous proteins and other biomolecules, including phospholipids (Silvius et al., 1987), to study the interaction of mRNA with the 30S ribosomal subunit (Czworkowski et al., 1991), in the investigation of cellular thiol components by flow cytometry (Durand and Olive, 1983), in the detection of carboxylate compounds using peroxyoxalate chemiluminescence (Grayeski and DeVasto, 1987), and for general sulfhydryl labeling (Sippel, 1981). 

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. 

We observed PKC-like activity in mouse forestomach extract (29). The activity was activated by TPA plus phosphatidylserine in the absence of calcium. Protein associated with this activity waspartially-puriEedby DEAE-cellulose chromatogrs hy. In mice pretreated with CLA 24 hours prior to sacrifice, the PKC-like activity was refractory to activation by TPA and phosphatidylserine. This observation indicates that cis-9, trans-11 incorporated into phospholipid might directly affect the interaction of TPA with PKC. Further, since PKC controls superoxide generation 30 CLA might in this way serve as an indirect antioxidant. 

In the bacterial PI-PLC structures, the top of the barrel rim has several hydrophobic residues that are fully exposed to solvent and poorly defined in the crystal structures (implying significant mobility). The active site of PI-PLC is accessible and well-hydrated, and these mobile elements at the top of the barrel offer a different motif for interactions of the protein with phospholipid interfaces. The PI-PLC from B. thuringiensis (nearly identical in sequence to the enzyme from B. cereus whose crystal structure was determined) exhibits the property of interfacial activation, where enhanced activity is observed when the substrate PI is present in an interface compared to monomeric substrate (Lewis et al., 1993). However, other non-substrate lipids such as phosphatidylcholine (PC), phosphatidic acid (PA), and other anionic lipids have an effect on the activity of PI-PLC toward both substrates PI and water-soluble cIP (Zhou et al., 1997). In particular, the presence of PC enhances the catalytic activity of... 

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