Cationic electrophoresis and electrotransfer of membrane glycoproteins

Sodium dodecyl sulfate electrophoresis of glycoproteins results in broad, fuzzy bands. The use of the positively charged detergent cetyltrimethylammonium bromide (CTAB) avoids this problem. Protocols for CTAB electrophoresis, electroblotting, and protein detection are presented. These methods were found useful in studies on the multiple drug resistance ATPase Mdr1.     

Intermediate filament-plasma membrane interactions.

In this review we will discuss the molecules involved in intermediate filament-desmosome and intermediate filament-hemidesmosome interactions, and the means by which certain of these molecules may bind to intermediate filaments. The possibility that intermediate filaments interact directly with peripheral membrane proteins and membrane lipids will also be addressed.     

Virus interactions with membrane filters

A survey of interactions of membrane filters with viruses has included 28 types of membranes, 4 types of enteroviruses, and 1 reovirus. Losses of these viruses in filtration, due to adsorption to the filter membranes, appear to be governed by three factors: the chemical composition of the filter membrane, the ratio of pore diameter to the diameter of the virus particle, and the presence of substances, such as those occurring in serum, which interfere with adsorption. Membranes of cellulose triacetate and of certain other materials have a very low affinity for these viruses. Cellulose triacetate filters adsorb virtually none when the pore size exceeds the virus diameter by a factor of more than 3. At porosities nearer the virus diameter, even low-affinity membranes adsorb large quantities of virus unless serum or some other additive interferes. Cellulose nitrate membranes, in the absence of interfering substances, adsorb enterovirus significantly at a pore size 285 times the virus diameter.     

Disruptive membrane interactions of alpha-synuclein aggregates

Alpha synuclein (αS) is a ~14 kDa intrinsically disordered protein. Decades of research have increased our knowledge on αS yet its physiological function remains largely elusive. The conversion of monomeric αS into oligomers and amyloid fibrils is believed to play a central role of the pathology of Parkinson's disease (PD). It is becoming increasingly clear that the interactions of αS with cellular membranes are important for both αS's functional and pathogenic actions. Therefore, understanding interactions of αS with membranes seems critical to uncover functional or pathological mechanisms. This review summarizes our current knowledge of how physicochemical properties of phospholipid membranes affect the binding and aggregation of αS species and gives an overview of how post-translational modifications and point mutations in αS affect phospholipid membrane binding and protein aggregation. We discuss the disruptive effects resulting from the interaction of αS aggregate species with membranes and highlight current approaches and hypotheses that seek to understand the pathogenic and/or protective role of αS in PD.     

Vesicle membrane interactions of penetratin analogues

Reports on serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides, also denoted protein transduction domains, have demonstrated the need for a reevaluation of the current understanding of peptide-mediated cellular delivery of large, hydrophilic molecules. In a recent study on the internalization in unfixed cells of penetratin and its analogues in which tryptophans are substituted for phenylalanines (Pen2W2F), lysines for arginines (PenArg), and arginines for lysines (PenLys), we revealed large dissimilarities in cell interactions among the peptides [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107]. We here investigated possible correlations with their respective affinities for the lipid membranes of large unilamellar vesicles. The variations found in membrane affinity correlated qualitatively with differences in hydrophobicity among the peptides but were by far too small to account for the striking differences in cell membrane binding. Interestingly, we found that the inclusion of a small fraction of lipids conjugated to poly(ethylene glycol) (PEG) in the vesicles both stabilized the vesicle dispersion against peptide-induced aggregation and, furthermore, enhanced the binding of the peptides to the membrane. By use of PEG-conjugated lipids, it could be shown that vesicle aggregation drives an alpha-helix to beta-sheet conformational transition for these peptides. A similar transition was discovered at submicellar concentrations of sodium dodecyl sulfate in aqueous solution for all peptides except PenLys. Finally, significant changes of the contributions to CD spectra from aromatic residues due to their insertion into the membrane were observed.     

Protein-protein interactions in intracellular membrane fusion

The fusion of intracellular vesicles with their target membranes is an essential feature of the compartmental structure of eukaryotic cells. This process requires proteins that dictate the targeting of a vesicle to the correct cellular location, mediate bilayer fusion and, in some systems, regulate the precise time at which fusion occurs. Recent biophysical and structural studies of these proteins have begun to provide a foundation for understanding their functions at a molecular level.     

Curvature-mediated interactions between membrane proteins.

Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions.     

alpha-Synuclein membrane interactions and lipid specificity

With the discovery of missense mutations (A53T and A30P) in alpha-synuclein (alpha-Syn) in several families with early onset familial Parkinson's disease, alpha-Syn aggregation and fibril formation have been thought to play a role in the pathogenesis of alpha-synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. As previous reports have suggested that alpha-Syn plays a role in lipid transport and synaptic membrane biogenesis, we investigated whether alpha-Syn binds to a specific lipid ligand using thin layer chromatography overlay and examined the changes in its secondary structure using circular dichroism spectroscopy. alpha-Syn was found to bind to acidic phospholipid vesicles and this binding was significantly augmented by the presence of phosphatidylethanolamine, a neutral phospholipid. We further examined the interaction of alpha-Syn with lipids by in situ atomic force microscopy. The association of soluble wild-type alpha-Syn with planar lipid bilayers resulted in extensive bilayer disruption and the formation of amorphous aggregates and small fibrils. The A53T mutant alpha-Syn disrupted the lipid bilayers in a similar fashion but at a slower rate. These results suggest that alpha-Syn membrane interactions are physiologically important and the lipid composition of the cellular membranes may affect these interactions in vivo.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)+cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS)+cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAPmss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) + cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP^mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Electric interactions at the lipid membrane surface

This work presents the results of an experimental study and of computer simulations concerning electric interactions in the surface layer of egg yolk lecithin (EYL) liposome membranes. The surface layer is formed by EYL polar heads, which possess features of electric dipoles, and positive charged polar heads belonging to admixtures of quaternary ammonium salts (AS). The results of the experimental study are in good agreement with the ones of the computer simulations. It was found that fluidity of the membranes, at a given concentration of AS, obtains the extremal (minimal) value. Similarly, the binding energy of the dipoles-positive charges system behaves like that in computer simulations. Moreover, the locations of the fluidity extremum and those of the binding energy depend on the charge of the AS polar heads as well as on the degree of electric interactions screening by the environment. At a certain optimal value of the screening coefficient, the energy of the system is the lowest (the most negative) and together with the rise in AS charge, the minimum of the energy moves towards its higher concentrations.     

Electrostatic interactions at the plasma membrane.

Hydrogen-ion titration has been used to detect the presence of charged groups on the human red-cell plasma membrane. The findings are discussed in terms of the effect of the local environment on electrostatic interactions between the charged groups.     

CapZ-lipid membrane interactions: a computer analysis

Background  

CapZ is a calcium-insensitive and lipid-dependent actin filament capping protein, the main function of which is to regulate the assembly of the actin cytoskeleton. CapZ is associated with membranes in cells and it is generally assumed that this interaction is mediated by polyphosphoinositides (PPI) particularly PIP₂, which has been characterized in vitro.     

Electrostatic interactions in an integral membrane protein

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.     

Model membrane platforms to study protein-membrane interactions

Abstract Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

Monopolar-bipolar lipid interactions in model membrane systems

1H-NMR, dynamic light scattering and negative staining electron microscopy have been used to study the formation and physico-chemical properties of aqueous dispersions of mixtures of monopolar lipids extracted from Sulfolobus solfataricus. This microorganism is a thermophilic archaeobacterium growing optimally at about 85 degrees C and pH 3. The two hydrolytic fractions of the membrane complex lipids that have been studied are: the symmetric lipid glycerol dialkyl glycerol tetraether (GDGT) and the asymmetric lipid glycerol dialkyl nonitol tetraether (GDNT). Electron micrographs of pure and mixed GDNT and GDGT dispersions show the formation of complex structures. Only above a critical monopolar/bipolar lipid ratio, typical of the bipolar lipid, could closed structures be formed and good agreement was obtained in sizing with NMR, electron microscopy and dynamic light scattering. NMR spectra have been carried out at several temperatures from 25 degrees to 85 degrees C, to obtain information on the temperature-dependent structural, dynamic and permeability properties of the co-dispersed vesicles. The results are discussed in terms of the steric constraints and the chemico-physical interactions occurring among the different parts of the molecules and compared with previous studies performed with different physical techniques.     

Model membrane platforms to study protein-membrane interactions

Abstract

Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.