Mutual effects of MinD-membrane interaction: I. Changes in the membrane properties induced by MinD binding

In Escherichia coli and other bacteria, MinD, along with MinE and MinC, rapidly oscillates from one pole of the cell to the other controlling the correct placement of the division septum. MinD binds to the membrane through its amphipathic C-terminal α-helix. This binding, promoted by ATP-induced dimerization, may be further enhanced by a consequent attraction of acidic phospholipids and formation of a stable proteolipid domain. In the context of this hypothesis we studied changes in dynamics of a model membrane caused by MinD binding using membrane-embedded fluorescent probes as reporters. A remarkable increase in membrane viscosity and order upon MinD binding to acidic phospholipids was evident from the pyrene and DPH fluorescence changes. This viscosity increase is cooperative with regards to the concentration of MinD-ATP, but not of the ADP form, indicative of dimerization. Moreover, similar changes in the membrane dynamics were demonstrated in the native inverted cytoplasmic membranes of E. coli, with a different depth effect. The mobility of pyrene-labeled phosphatidylglycerol indicated formation of acidic phospholipid-enriched domains in a mixed acidic-zwitterionic membrane at specific MinD/phospholipid ratios. A comparison between MinD from E. coli and Neisseria gonorrhea is also presented.     

Mutual effects of MinD-membrane interaction: II. Domain structure of the membrane enhances MinD binding

MinD, a well-conserved bacterial amphitropic protein involved in spatial regulation of cell division, has a typical feature of reversible binding to the membrane. MinD shows a clear preference for acidic phospholipids organized into lipid domains in bacterial membrane. We have shown that binding of MinD may change the dynamics of model and native membranes (see accompanying paper [1]). On the other hand, MinD dimerization and anchoring could be enhanced on pre-existing anionic phospholipid domains. We have tested MinD binding to model membranes in which acidic and zwitterionic phospholipids are either well-mixed or segregated to phase domains. The phase separation was achieved in binary mixtures of 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG) with 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) or 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) and binding to these membranes was compared with that to a fluid mixture of SOPG with 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC). The results demonstrate that MinD binding to the membrane is enhanced by segregation of anionic phospholipids to fluid domains in a gel-phase environment and, moreover, the protein stabilizes such domains. This suggests that an uneven binding of MinD to the heterogeneous native membrane is possible, leading to formation of a lipid-specific distribution pattern of MinD and/or modulation of its temporal behavior.     

Effects of small nonpolar molecules on membrane compressibility and permeability: A theoretical study of the effects of anesthetic gases

We explore from a theoretical perspective the effects of small nonpolar molecules, such as anesthetic gases, on membrane compressibility and permeability. As a model system we expand a previously proposed generalization of Nagle's model for biomembrane phase transitions. In this model anesthetic gases alter membrane compressibility, causing profound changes in membrane permeability. Anesthetics either increase or decrease membrane permeability, depending on whether the membrane lipid is originally in the solid or melted state, or in a two-phase region. These changes are reversed by high pressure, in agreement with experimental results. Anesthetic-induced changes in compressibility are predicted to inhibit fusion of phospholipid vesicles to each other and to planar bilayers, and thus might be expected to inhibit the fusion of presynaptic vesicles with the presynaptic nerve membrane. This work provides a detailed molecular theory for many of the effects of anesthetic gases on both synapse and axon, and provides a coherent framework for understanding diverse experimental results.     

Prediction of the most favorable configuration in the ACBP-membrane interaction based on electrostatic calculations

Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP-membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding (dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP-membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.     

CTP:phosphocholine cytidylyltransferase: Function,regulation, and structure of an amphitropic enzyme required for membrane biogenesis

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-limiting and regulated step in the CDP-choline pathway for the synthesis of phosphatidylcholine (PC) and PC-derived lipids. Control of CCT activity is multi-layered, and includes direct regulation by reversible membrane binding involving a built-in lipid compositional sensor. Thus CCT contributes to phospholipid compositional homeostasis. CCT also modifies the curvature of its target membrane. Knowledge of CCT structure and regulation of its catalytic function are relatively advanced compared to many lipid metabolic enzymes, and are reviewed in detail. Recently the genetic origins of two human developmental and lipogenesis disorders have been traced to mutations in the gene for CCTα.     

A 1H-NMR investigation of the effects of ethanol and general anaesthetics on ion channels and membrane fusion using unilamellar phospholipid membranes

Using ¹H-NMR of small unilamellar vesicles in the presence of the lanthanide probe ion Pr³⁺, the effects of ethanol, diethyl ether and chloroform on various mechanisms of channel-mediated transport were studied. The mechanisms include channel formation by the polypeptide Alamethicin 30 and vesicular lysis at the gel to liquid-crystal phase transition of the lipid. Channel stabilisation and membrane fusion induced by sub-critical micelle concentrations of Triton X-100 were also investigated. The observation that ethanol and diethyl ether increase membrane permeability and fusion while chloroform inhibits them suggests a common locus of action on the properties and structure of channel-associated water. This conclusion is discussed in terms of current theories of general anaesthesia.     

A simple theoretical model for the effects of cholesterol and polypeptides on lipid membranes

A simple theoretical model for the effects of impurities on biomembranes is proposed. The model accounts for the cholesterol-induced decrease of membrane phase transition temperature, membrane condensation above the gel to liquid crystalline phase transition, and increase in lateral compressibility. The model also predicts that addition of molecules such as cholesterol and polypeptides to membranes results in unmasking of a continuous phase transition. This results in a second broad peak in the calorimetric curves for melting of lipid-cholesterol mixtures, and the appearance of a second melting transition in membranes modified by the incorporation of polypeptides. The theory assumes that the membrane may be adequately described by a kink model, and that impurities are randomly distributed in the membrane. The difference in size and shape of impurity molecules, compared to membrane lipids, results in a spatial disordering in the membrane which in turn causes increased chain disorder and membrane condensation, as well as a decrease in the cooperativity of melting. The second transition results from a second expansion of the condensed, partially disordered membrane, which takes place over a several degree temperature range. This transition, although unmasked by boundary effects of non-lipid molecules, does not correspond to melting of a boundary annulus or phase separation.     

Effects of n-Alkanols on the membrane fluidity of chick embryo heart microsomes

The $\text{n-}\text{alkanols}$ from butanol through octanol are membrane perturbing agents that fluidize the microsomal membranes of 20-day-old chick embryo hearts as measured by the fluorescence depolarization of 1,6-diphenylhexatriene. In terms of the aqueous concentrations of $\text{n-}\text{alkanols}$ the fluidizing effect increases with increasing number of carbons per $\text{n-}\text{alkanol}$. In terms of the membrane concentrations of $\text{n-}\text{alkanols}$ the fluidizing effect is roughly equivalent for all the $\text{n-}\text{alkanols}$ studied.     

Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles Effect of membrane composition and temperature

Small unilamellar vesicles were used to measure the permeability of saturated phosphatidylcholine bilayers to glucose. The presented method circumvents most of the common restrictions of classical permeability experiments. Increasing the fatty acid chain length of the lipids reduced the permeation rate significantly. Raising the temperature above that of the lipid phase transition drastically increased membrane permeability. Arrhenius plots demonstrated the activation energy to be independent of membrane composition and the phase-state of the lipids. The permeation process is discussed in terms of a constant energy to disrupt all hydrogen bonds between permeant and aqueous solvent prior to penetrating the membrane. The magnitude of the permeability coefficient is partly determined by a unfavourable change in entropy of activation on crossing the water/lipid interface. All results indicate that the penetration of the dehydrated permeant into the hydrophobic barrier is the rate-limiting step in the permeation of glucose.     

Automated on-like dialysis, trace enrichment and high-performance liquid chromatography Inhibition of interaction with the dialysis membrane and disruption of protein binding in the determination of clozapine in human plasma

Problems related to interaction of drugs with the dialysis membrane and to protein binding must be overcome in order to develop automated methods for drug analysis based on on-line dialysis, trace enrichment and HPLC. In order to study these problems, clozapine and its active metabolite N-desmethylclozapine were chosen as model compounds because they were found to interact with the dialysis membrane, and clozapine is highly protein bound. Addition of a cationic surfactant, dodecylethyldimethyl ammonium bromide, to the donor solution and to the plasma samples was found to inhibit interaction of the drugs with surfaces. The protein binding in plasma was disrupted prior to dialysis by lowering the pH with hydrochloric acid and the plasma proteins were solubilised with glycerol. The results obtained were used to develop a fully automated method for the determination of clozapine and N-desmethylclozapine in human plasma. More than 100 samples could be analysed within 24 h. The limit of detection in human plasma was 0.050 μmol/1 for clozapine and 0.055 μmol/1 for N-desmethylclozapine. Linearity was found for drug concentrations between 0.25–3 μmol/1. The relative standard deviations were between 1.2–6.7% and the method was applicable for therapeutic drug monitoring.     

Chiral interaction in peptide molecules: effects of chiral peptide species on helix-sense induction in an N-terminal-free achiral peptide

Noncovalent chiral domino effect (NCDE) has been proposed as terminal-specific interaction upon a 3(10)-helical peptide chain, of which the helix sense is manipulated by an external chiral stimulus (mainly amino acid derivatives) operating on the N-terminus (Inai, Y., et al. J Am Chem Soc 2000, 122, 11731-11732; ibid., 2002, 124, 2466-2473; ibid., 2003, 125, 8151-8162). We have investigated here a helix-sense induction in an optically inactive N-terminal-free nonapeptide (1) through the screening of several peptide species that differ in chiral sequence, chain length, and C-terminal group. Helix-sense induction in peptide 1 depends largely on both the C-terminal chirality and carboxyl group in the external peptide, in which N-carbonyl-blocked amino acids, "monopeptide acids," should be the minimum requirement for effective induction. N-Protected mono- to tetrapeptides of L-Leu residue commonly induce a right-handed helix. NMR study and theoretical computation reveal that the N-terminal segment of peptide 1 binds the N-protected dipeptide molecule through multipoint coordination to induce a right-handed helix preferentially. The present findings not only will improve our understanding of the chiral roles in peptide or protein helical termini, but also might demonstrate potential applications to chirality-responsive materials based on peptide helical fragments.     

Membrane-SPINE: an improved method to identify protein-protein interaction partners of membrane proteins in vivo

Membrane proteins are crucial for many essential cellular processes. As membrane proteins function in complexes, methods to detect and to characterize membrane protein-protein interactions are undoubtedly required. Therefore, we developed the "Membrane-Strep-tagged protein interaction experiment" (Membrane-SPINE) that combines the specific purification of a Strep-tagged membrane protein with the reversible fixation of protein complexes by formaldehyde cross-linking. In combination with MS analysis, we suggest Membrane-SPINE as a powerful tool to identify unknown interaction partners of membrane proteins in vivo.     

The interaction of versican with its binding partners

Versican belongs to the family of the large aggregating chondroitin sulfate proteoglycans located primarily within the extracellular matrix （ECM）. Versican, like other members of its family, has unique N- and C-terminal globular regions, each with multiple motifs. A large glycosaminoglycan-binding region lies between them. This review will begin by outlining these structures, in the context of ECM proteoglycans. The diverse binding partners afforded to versican by virtue of its modular design will then be examined. These include ECM components, such as hyaluronan, type Ⅰ collagen, tenascin-R, fibulin-1, and -2, fibrillin-1, fibronectin, P- and L-selectins, and chemokines. Versican also binds to the cell surface proteins CD44, integrin β1, epidermal growth factor receptor, and P-selectin glycoprotein ligand-1. These multiple interactors play important roles in cell behaviour, and the roles of versican in modulating such processes are discussed.     

Modulation of prolactin binding sites in vitro by membrane fluidizers II. Age-dependent effects on rat ventral prostatic membranes

The objectives of this study were to determine (i) if the age-related changes in ¹²⁵I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24–25 days old), young-adult (80–90 days old) and aged (550–610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.     

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.     

NMR solution structure of the N-terminal domain of hERG and its interaction with the S4-S5 linker

The human Ether-à-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation. To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.     

Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus K _c of the membrane and of the cytoplasmic viscosity were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I ₀ passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of K _c /R _inf0 ^sup3 and · R ₀ (R ₀=equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of K _c = 2–3 · 10^–19 Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, K _c , = 3.4 · 10^–20 Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (K _c = 5 · 10^–19 Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent K _c and values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocytestomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCI solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) K _c and are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent K _c and after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance. (2) Shape transitions may be caused by relative area changes either of the two monolayers of the lipid/protein bilayer (corresponding to the bilayer coupling hypothesis) or of the bilayer and the cytoskeleton where the latter mechanism appears to be more frequent. (3) The low bending stiffness and the shear free deformation regime are explained in terms of a slight excess area of the lipid bilayer leading to a pre-undulated surface profile. Freeze fracture electron microscopy studies provide direct evidence for a pre-undulated bilayer with an undulation wavelength of approximately 100 nm. Offprint requests to: E. Sackmann     

Mechanism of interaction of PITPalpha with membranes: conformational changes in the C-terminus associated with membrane binding

Eukaryotic phosphatidylinositol transfer proteins (PITPs) are composed predominantly of small ( approximately 32 kDa) soluble proteins that bind and transfer a single phospholipid, normally phosphatidylinositol or phosphatidycholine. Two forms, PITPalpha and PITPbeta, which share approximately 80% amino acid sequence similarity, are known. Rat PITPalpha was labeled at specific single reactive Cys residues with I-AEDANS and used to examine PITP-membrane interactions. Upon binding to phospholipid vesicles, PITP labeled with AEDANS at the C-terminus, a region postulated to be involved in membrane binding, shows significant decreases in both steady-state and dynamic fluorescence anisotropy. In contrast, PITPs labeled with AEDANS at sites located distal to the C-terminus show increases in both steady-state and dynamic anisotropy. These results suggest that interaction of PITP with membrane surfaces leads to significant alterations in conformation and perhaps melting of the C-terminal helix.