Reconstitution in lipid vesicles

D.Y. Kim, Y. Kam, S.K. Koo, C.O. Joe, Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation, J Biol Chem 274, 5581-5587 (1999). 

We have recently used polarized infrared (IR) spectroscopy to examine the extent and organization of protein structures in membrane associated proteins (4-6). In particular, with membrane reconstituted RC, we "ave determined the net orientation of thea -helical segments with respect to the membrane plane (6). In this article, we have made a comparison of the protein structures in the LMH and LM complexes in order to analyze the composition and the orientation of the secondary structures localized within the LM complex. We have investigated the UV circular di-chroism (CD) and polarized IR spectra of the LMH and LM complexes reconstituted in lipid vesicles. Our results show that transmembrane a-helices are present in both LMH and LM complexes. In addition, we also discussed some general features of membrane IR dichroism spectra. 

One criterion for a bona fide membrane-fusion protein is that membrane fusion can be reconstituted by transfection of the candidate fusion protein into nonfusing cells or by reconstitution into lipid vesicles (White, 1990 White, 1992 White and Blobel, 1989). Transfection of meltrin a into fibroblasts did not lead to an increase in cell fusion (Yagami-Hiromasa et al., 1995). Clearly, failure to reconstitute fusion does not rule out a role in fusion because the cellular fusion machinery may be more complex than viral fusion proteins. Muscle cells might contain other proteins that are required for meltrin a to promote membrane fusion but that are not expressed or active in fibroblasts. Ultimately, the identity of a bona fide cell-cell fusion protein or protein machine will only be determined by reconstituting membrane fusion with defined components. In the interim, it will be important to more accurately understand the roles of meltrin a and fertilin in the cascade of steps that result in membrane fusion and thereby perhaps distinguish between a direct and indirect role in fusion. 

Relatively little is known about the physical properties of ACAT. Enzyme activity has been solubilized from different tissues and reconstituted into lipid vesicles, but has been purified only partially [24,25]. Even the kinetics and mechanism of the ACAT reaction are poorly understood. As in the case of most enzymes involved in lipid metabolism, kinetic constants are difficult to determine because the substrates are water insoluble and membrane associated [26]. Moreover, systematic efforts to identify the active site of the enzyme through the use of active site-directed reagents have yet to be made. Such efforts might be worthwhile even at this stage of investigation of the enzyme because the use of radioactive, site-directed reagents might allow identification of the enzyme protein. 

R. J. Lefkowitz, Reconstitution of beta-adrenergic receptors in lipid vesicles affinity... 

The crystals discussed above contain only about 30% protein and about 70% water, detergent, and crystallization buffer (1), causing the signal/noise ratio of the difference spectra to decrease as compared with simpler protein systems. Reaction centers reconstituted into lipid vesicles contain more protein in comparison to water, buffer, and lipid and should provide a higher signal/noise ratio. Thus the reconstituted system should increase the probability of seeing small changes in the difference spectra. 

The universal character of calorimetric measurements also pays in the elucidation of the reversible transformations undergone by bilayer-forming phospholipids. The transitions of phosphatidylcholine and similar congeners between their vesicular and micellar states depending on their concentrations, the presence of simple detergents, and the temperature are quite sharp and accompanied by sensible heat effects that allow for their thermodynamic characterization. In a particularly illustrative example, the dissolution and reconstitution of lipid vesicles from Escherichia coli native polar lipid fraction by octyl-jS-o-glucoside as analyzed by ITC was reported (Figure 15). ... 

As shown in Table 4, in both PM preparations, vanadate-sensitive ATPase (pH 6.5) exhibits similar properties and hydrolyzes ATP to the same extent. The thermotropic behavior of the enzyme as evidenced by Arrhenius plots was also found to be similar in both fractions (data not shown), thus ruling out any important contribution by the modification of the sterol profile of the PM to ATP hydrolysis. Some preliminary experiments were carried out with an ATPase reconstituted into lipid vesicles containing or not sterols (A -sterols or 24-methylpollinastanol). H -pumpinq was measured by using the fluorescent probe quinacrine. Sterols were found to induce a significant inhibition (55 to 65%) of H -transport compared to that measured in the absence of sterols. By contrast, only low changes (10%) in ATP hydrolysis were observed by addition of sterols in liposomes. 

The crystallization [1] and subsequent x-ray diffraction analysis [2,3] of the reaction center from Rps, viridis have provided a detailed picture of the chromophores in their ground states, PIQaQb The crystals exhibit linear dichroism [4,5] and have been shown to be photoactive [4], A series of experiments was therefore undertaken to investigate various charge-separated states stabilized by continuous illumination (1) difference Fourier analysis of x-ray diffraction data sets for illuminated and dark crystals was performed for reaction centers reconstituted with ubiquinone-9, yielding differences between P QaQb and PQaQb/ and (2) light-induced FTIR difference spectra were measured for crystals and for reaction centers reconstituted into lipid vesicles to identify differences between Qa and Qb. Furthermore, using reconstituted reaction center samples it was possible to selectively stabilize P Qa P Qb"/ PI / PI Qa / and PI Qa. Thus the electron transfer pathway could be followed from the primary donor, P, to the secondary acceptor, Qb, via the intermediary (bacteriopheophytin) acceptor, I, and Qa. 

Effect of lipid phase transition on the relative ampiitude-of the fast component of P charge recombination in Rps. viridis reaction centers reconstituted in DMPC vesicles... 

Dufour, J. R Goffeau, A. Tsong, T. Y. Active proton uptake in lipid vesicles reconstituted with the purified yeast plasma membrane ATPase. Eluorescence quenching of 9-amino-6-chloro-2-methoxyacridine. J. Biol. Chem. 1982, 257, 9365-9371. 

Cell membranes or synthetic lipid vesicles with normal low permeability to water will, if reconstituted with AQP1, absorb water, swell and burst upon exposure to hypo-osmotic solutions. The water permeability of membranes containing AQP 1 can be about 100 times greater than that of membranes without aquaporins. The water permeability conferred by AQP1 (about 3 billion water molecules per subunit per second) is reversibly inhibited by Hg2+, exhibits low activation energy and is not accompanied by ionic currents or translocation of any other solutes, ions or protons. Thus, the movement of water through aquaporins is an example of facilitated diffusion, in this case driven by osmotic gradients. 

Most attractive are solid-state NMR studies of membrane proteins, in their native membranes, or membrane proteins reconstituted into synthetic lipid vesicles with defined lipid composition. Several integral membrane proteins are present in sufficiently high concentrations in the native membranes (> 25% of total membrane protein) to offer samples concentrated enough to permit the application of solid-state NMR without... 

Fig. 52a-c. Scheme of the fusion process of giant liposomes and the formation of small unilamellar vesicles (SUV) at the interface, a) lipid bilayers in contact b) pores generated by electric breakdown and lipid reorientation forming SUVs c) reconstitution of lipid membranes formation of a fused giant liposome and SUVs . 

ATPase reconstituted in vesicles of different lipid composition [26], Pumping efficiency (number of Ca2+ ions transported per molecule of ATP hydrolyzed) increased with the mole fraction of lipids that prefer Hn-phase formation. However, no such correlation was observed with dioleoylphosphatidylcholine or digalactosyldiglyc-eride, which do not form low-temperature H n phases. It is important to note that it is not the chemical similarity but the phase preference of the phospholipid that is decisive. 

A number of factors influence formation of proteoliposomes with a low passive ion permeability. Lin et al. (1990) have reported that polyethylene glycol (PEG) treatment of proteoliposomes lowers passive permeability to small molecules, enabling the ion-gradient to be maintained for longer periods. In several ion-coupled carrier reconstitution procedures (Fafoumoux et al., 1989 Tamarappoo and Kilberg, 1991 Ramamoorthy et al., 1992 Ramamoorthy et al., 1993), solubilized proteins have been precipitated with PEG before incoiporation into lipid vesicles. Trace amounts of PEG associated with the proteoliposomes in these procedures may reduce passive permeability and lead to high levels of ion-coupled transport (Ramamoorthy et al., 1993). 

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