Protein system, lipid

Our studies of the interaction effects have already shown that valuable information can be obtained about the mode of interaction of these molecules within these membrane fragments. NMR spectroscopy appears sufficiently promising as a technique for us to expand our studies to other nuclei, such as phosphorus and nitrogen, and also to other biological membranes and lipid-protein systems. In this way we hope to provide increased understanding of the way in which the constituents of membranes are associated. A more complete account of our NMR studies of membranes will be published (4). 

Besides the asymmetry between monolayers in cytomembranes, two of the more obvious differences between cubic phases and membranes are the unit cell size and the water activity. It has been argued that tire latter must control the topology of the cubic membranes [15], and hence tiiat the cubic membrane structures must be of the reversed type (in the accepted nomenclature of equilibrium phase behaviour discussed in Chapters 4 and 5 type II) rather than normal (type I). All known lipid-water and lipid-protein-water systems that exhibit phases in equilibrium with excess water are of the reversed type. Thus, water activity alone cannot determine the topology of cubic membranes. Cubic phases have recently been observed with very high water activity (75-90 wt.%), in mixtures of lipids [127], in lipid-protein systems [56], in lipid-poloxamer systems [128], and in lipid A and similar lipopolysaccharides [129,130]. 

Information about fluidity and viscosity of bilayers of artificial and natural membranes has been obtained from electron spin resonance studies in which the mobility of the spin-labelled species along the surface plane of the membrane is determined (17). However, the monolayer of either lipid, protein, or lipid-protein systems at the air-water interface, makes an ideal model because several parameters can be measured simultaneously. Surface tension, surface pressure, surface potential, surface viscosity, surface fluorescence and microviscosities, surface radioactivity, and spectroscopy may be determined on the same film. Moreover, the films can be picked up on grids from which they may be observed by electron microscopy, studied further for composition, and analyzed for structure by x-ray diffraction and spectroscopy. This approach can provide a clear understanding of the function and morphology of the lipid and lipid-protein surfaces of experimental membranes. However, the first objective is to obtain molecular correlations of surface tension, pressure, potential, and viscosity. 

Protein and Lipid-Protein Systems. The high surface viscosity of BSA and the low surface viscosity of RNase had been observed by a torsion rotational method that used an extremely large torque (2, 6). In the present experiments, the protein is dispersed in the aqueous subphase to a final concentration of 10 /xg/ml. Both film pressure and surface viscosity are measured as a function of time (to 40 min). The film pressure of BSA was 21 dynes/cm at mixing and remained constant for 40 min. Also, surface viscosity reached a high value instantaneously (although the first measurement was made at 5 min) and remained constant for 40 min. In contrast, RNase built up pressure slowly, from 3 dynes/cm... 

Lipid—Lipid and Lipid—Protein Interactions. The DPL—cholesterol and the protein—DPL systems are particularly amenable to interpretation using our membrane model. The high viscosity lattice of DPL can be broken by cholesterol (Figure 9), and the lattice of BSA can be broken by a lipid (e.g., DPL, Figure 10), with a marked loss of surface viscosity. This lattice collapse means formation of independent membrane subunits whose lateral valences are saturated within the subunit, thereby producing a fluid system (Figure 1A) the subunit could be a lipid-lipid system, as with DPL and cholesterol, or a lipid-protein system. The phenomenon of lattice collapse with loss of surface viscosity is impressive in the DPL-albumin system since individually both components have a high surface viscosity. 

The results presented in Table 1 for the n-alcohols are all based on interactions with lipid-protein systems. Results on lipid systems only, show a similar trend. Table T summarizes a number of these studies. The Ay value for the data from Table T is -858 cal/mole with a standard deviation of 221. This value is very similar to the overall value for the lipid-protein systems (Table 6). 

In both approaches a series of equilibration steps based on energy minimization is used to obtain the final configuration of the lipid/protein system. In the latter case, the process does not affect the initial lipid configuration significantly outside the cavity region, and the final configuration of the lipid/... 

As the reflectivity curve R qz) does not contain enough information to deduce the positions, Zj of the individual atoms, this strategy requires that an atomic model can be generated and parametrically varied for agreement with the observed R qz) this can be cumbersome for large molecules such as proteins or lipid-protein systems The model should include all atoms or molecules contributing to the electron density also, e.g., bound water molecules. 

First the timescale of lipid translation and rotation is typically much longer than the length of the simulation. This makes the initial conditions for a lipid-protein system of extreme importance since in the course of several nanoseconds not much large-scale rearrangement can occur. How does one prepare a bilayer system with a transmembrane helix without predetermining the outcome of the simulation by the initial conditions ... 

Table 3.1. Stoichiometries of the Motionally-Restricted Lipid Spin Label Component in Various Lipid-Protein Systems...

Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC) have become standard techniques in the field of thermodynamic investigation of natural membranes and model membrane systems. Due to the new developments of more sensitive DSC instruments, studies of lipid-protein systems liave become feasible, which could previously not be perfonued because of the limitations on amount of material. It is to be expected that DSC methods will again make a large progress because of these improvements in sensitivity. 

To use liposomes as delivery systems, drug is added during the formation process. Flydrophilic compounds usually reside in the aqueous portion of the vesicle, whereas hydrophobic species tend to remain in the lipid proteins. The physical characteristics and stability of lipsomal preparations depend on pH, ionic strength, the presence of divalent cations, and the nature of the phospholipids and additives used [45 47],... 

IVFE 10% and 20% products can be administered by a central or peripheral vein, added directly to PN solution as a total nutrient admixture (TNA) or three-in-one system (lipids, protein, glucose, and additives), or piggybacked with a CAA and dextrose solution. IVFE 30% is approved only for TNA preparation. 

Lipidated peptides embodying the characteristic linkage region found in the parent lipoproteins and bearing additional functional groups, which could be traced in biological systems or which allowed for their use in biophysical experiments, were used successfully in model studies. However, such model studies only provide a limited amount of information. In order to approximate the situation in a biological system more precisely, experiments with differently lipidated proteins are required. 

Studying the sequences of farnesylated proteins indicated that all lipidated proteins bear a cysteine residue near the C-terminus revealing the CAAX-motif, where C is a cysteine, A stands for an aliphatic amino acid, and X can be any amino acid. Database searches resulted in more prenylated proteins, all bearing the CAAX-motif, in systems from lower eukaryotes to mammals. A closer look at the mature proteins revealed that prenylation was only the first step of processing of the CAAX-motif-encoded proteins. After transfer of the isoprene unit, the last three amino acids are cleaved proteolytically by an endoprotease and the C-terminal cysteine is carboxymethylated by a methyltransferase. ... 

Post-translational modifications, such as phosphorylation, complex glycosylation, and lipidation, typically occur in eukaryotic organisms. Therefore, their expression in prokaryotic systems like Escherichia coli is difficult. However, it should be noted that via clever engineering and coexpression of specific enzymes, access can be granted to specific lipidated proteins via expression in bacteria, for example, via the expression of A -myristoyltransferase in E. coli Eukaryotic systems that can be used for the expression of post-translationally modified proteins are yeast and Dictyostelium discoidum. Furthermore, lipidated proteins, such as the Rah proteins, can be obtained via purification from tissue sources or from membrane fractions of insect cells that had been infected with baculovirus bearing a Rah gene. ... 

Our work deals with the necessity of creating kinetics laws for heterogeneous enzymology. There was a big gap between the classical enzyme kinetics in solution and highly structured biological systems. All the concepts of diffusion reaction are clear for our thick membrane but are also useful for lipid-protein membranes, even if the process of transport is not only classical diffusion. 

T,he stoichiometric characterization of detergent-protein complexes has been the object of many studies over the past 30 years (6). Recent studies have placed more emphasis upon developing a molecular-kinetic description of the complex (2, 8). The importance of such descriptions lies in the fact that detergent-protein complexes can be considered as lipoprotein model systems. Indeed, virtually all conceptions of the microscopic nature of lipid-protein interactions are based on the properties of detergent-protein complexes (3). 

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