The effect of ATP on the cell membrane structure]

ATP influence on the structure of plasma membranes thymocytes of cattle was studied. Fluorescence anisotropy of tryptophan residues of membrane proteins, fluorescence anisotropy of 3-methoxybenzanetron and fluorescence intensity of 1-anilinonaphthalene-8-sulphonate were determined. Changes of tryptophan fluorescence anisotropy and of ANS fluorescence intensity were established. It is supposed that the observed changes are connected with the change of membrane proteins structure and plasma membrane charge.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

Polarization-optical investigation (topo-optical analysis) of the structure of the human erythrocyte glycocalyx. Influence of pH, ionic strength and diamide-induced spectrin cross linking

The value of membrane anisotropy after fixation and topo-optical analysis of erythrocytes stained with toluidine blue is a measure for the degree of spatial order of the dyestuff-binding acidic residues of the glycocalyx and thus a parameter for the characterization of the glycocalyx structure. Lowering the pH value and/or the ionic strength of the staining medium results in a decrease of membrane anisotropy indicating a lower order of the anionic residues. In agreement with the findings of other authors this phenomenon seems to be connected with an expansion of the glycocalyx. Diamide-induced oxidative crosslinking of spectrin before fixation and staining with toluidine blue at physiological pH and ionic strength also results in a decreased anisotropy. This indirect influence on the glycocalyx structure may be caused by an increase of the charge density within the glycocalyx due to a diamide-induced rearrangement of the membrane skeleton and of the transmembrane proteins bound to it.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.     

Interplay between the electrostatic membrane potential and conformational changes in membrane proteins

Transmembrane electrostatic membrane potential is a major energy source of the cell. Importantly, it determines the structure as well as function of charge‐carrying membrane proteins. Here, we discuss the relationship between membrane potential and membrane proteins, in particular whether the conformation of these proteins is integrally connected to the membrane potential. Together, these concepts provide a framework for rationalizing the types of conformational changes that have been observed in membrane proteins and for better understanding the electrostatic effects of the membrane potential on both reversible as well as unidirectional dynamic processes of membrane proteins.     

Interaction of clathrin coat proteins with unilamellar and multilamellar vesicles of phosphatidylcholine

The binding of clathrin and accessory coat proteins to small unilamellar vesicles and to liposomes of uncharged phospholipids has been followed by chromatography, 31P-NMR, ESR and fluorescence anisotropy. At pH 6.5 and at an ionic strength value (0.1 M Mes) close to that used during the purification of clathrin-coated vesicles, the proteins do not restore the characteristic network found around the natural vesicles. Instead, a limited fusion leads to enlarged structures in which the perturbation of the dynamics of the phospholipids decreases gradually with the depth in the membrane. While the rate of motion of the outer polar heads is lowered, the order parameter of doxyl groups located either under or in the vicinity of the glycerol backbone is not affected by the proteins. In the inner core of the membrane, the main thermotropic transition of the hydrocarbon chains is unchanged. All the effects are the results of interactions limited to the membrane surface. The electrostatic nature of these interactions is evidenced when the embedded spin labels have a charge protruding at the membrane surface. An 'anchoring' effect appears which is due to the charged groups of the proteins. The lateral diffusion of the probes is reduced and, at low ionic strength, a cationic derivative no longer detects the thermotropic transition of the hydrocarbon chains. These results indicate that, although it is known that clathrin and accessory proteins bind to membranes by a series of protein-protein interactions, this system is not devoid of lipid-protein interactions, at least when it is not organized as in the natural system.     

Specific Xray diffraction patterns of membrane proteins caused by secondary structure collinearity

Diffraction anisotropy is a phenomenon that impacts more specifically membrane proteins, compared to soluble ones, but the reasons for this discrepancy remained unclear. Often, it is referred to a difference in resolution limits between highest and lowest diffraction limits as a signature for anisotropy. We show in this article that there is no single correlation between anisotropy and difference in resolution limits, with notably a substantial number of structures displaying various anisotropy with no difference in resolution limits. We further investigated diffraction intensity profiles, and observed a peak centred on 4.9 Å resolution more predominant in membrane proteins. Since this peak is in the region corresponding to secondary structures, we investigated the influence of secondary structure ratio. We showed that secondary structure content has little influence on this profile, while secondary structure collinearity in membrane proteins correlate with a stronger peak. Finally, we could further show that the presence of this peak is linked to higher diffraction anisotropy. These results bring to light a specific diffraction of membrane protein crystals, which calls for a specific handling by crystallographic software. It also brings an explanation for investigators struggling with their anisotropic data.     

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.     

Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal brush-border membranes

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.     

Membrane order and molecular dynamics associated with IgE receptor cross-linking in mast cells

Cholesterol-rich microdomains (or "lipid rafts") within the plasma membrane have been hypothesized to exist in a liquid-ordered phase and play functionally important roles in cell signaling; however, these microdomains defy detection using conventional imaging. To visualize domains and relate their nanostructure and dynamics to mast cell signaling, we use two-photon (760 nm and 960 nm) fluorescence lifetime imaging microscopy and fluorescence polarization anisotropy imaging, with comparative one-photon anisotropy imaging and single-point lifetime and anisotropy decay measurements. The inherent sensitivity of ultrafast excited-state dynamics and rotational diffusion to the immediate surroundings of a fluorophore allows for real-time monitoring of membrane structure and organization. When the high affinity receptor for IgE (FcepsilonRI) is extensively cross-linked with anti-IgE, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids (the lipid analog diI-C(18) or glycosphingolipids)) and lipid-anchored proteins coredistribute with cross-linked IgE-FcepsilonRI. We find an enhancement in fluorescence lifetime and anisotropy of diI-C(18) and Alexa 488-labeled IgE-FcepsilonRI in the domains where these molecules colocalize. Our results suggest that fluorescence lifetime and, particularly, anisotropy permit us to correlate the recruitment of lipid molecules into more ordered domains that serve as platforms for IgE-mediated signaling.     

N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology.

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.     

Effect of calcium ions on plasma membrane structure

The structure of thymocyte's plasma membranes was studied as affected by calcium ions (0-1.126 mM). The fluorescence intensity of 1-anilinonaphthalene-8-sulphonate, the epimerization degree of pyrene and fluorescence anisotropy of membrane proteins were studied. The change of electrochemical properties of membranes, conformation of membrane proteins and lipid fluidity has been shown.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

Cysteine-rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants

Two cysteine-rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58,000 (outer membrane protein 2; OMP2) and 15,000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.     

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381-410; (1985) Biochim. Biophys. Acta 812, 361-368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.     

Structure determination of membrane proteins by NMR spectroscopy.

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.     

Energization-induced redistribution of charge carriers near membranes

The electric field arising from proton pumping across a topologically closed biological membrane causes accumulation close to the membrane of ionic charges equivalent to the charge of the pumped protons, positive on the side towards which protons are pumped, negative on the other side. We shall call this the 'active surface charge'. We here use the Poisson-Boltzmann equation to evaluate the effects of zwitterionic buffer molecules and uncharged proteins in the aqueous phase bordering the membrane on the magnitude and ionic composition of the active surface charge. For the positive side of the membrane, the main results are: (1) If the membrane is freely accessible to bulk phase ions, pumped protons exchange with these ions, such that the active surface charge consists of salt cations. (2) If a significant fraction of the ions in bulk solution consists of buffer molecules, then some of the pumped protons will remain close to the membrane and constitute a major fraction of the active surface charge. (3) If a protein layer borders the membrane, a significant part of the transmembrane electric potential difference exists within that protein layer and protons inside this layer dominate the active surface charge. (4) On the negative side of the membrane the corresponding phenomena would occur. (5) All these effects are strictly dependent on the transmembrane electric potential difference arising from proton pumping and would come in addition to the well known effects of buffers and electrically charged proteins on the retention of scalar protons. (6) No additional proton diffusion barrier may be required to account for a deficit in number of protons observed in the aqueous bulk phase upon aeration-induced proton pumping.