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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Biochim Biophys Acta. 2008 Aug 12;1778(11):2505–2511. doi: 10.1016/j.bbamem.2008.08.004

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

Shirley Mazor 1, Tomer Regev 1, Eugenia Mileykovskaya 2, William Margolin 3, William Dowhan 2, Itzhak Fishov 1,*
PMCID: PMC2592533  NIHMSID: NIHMS79405  PMID: 18760260

Summary

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 preexisting 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.

Keywords: membrane domain, protein-membrane interaction, α helix, phase transition

Introduction

Spatial control of division in a bacterial cell operated by the Min system is based on prevention of FtsZ ring formation in nucleoid-free regions of the cell, e.g. on poles of rod-shaped cells (for a review see [2]). This implies a polar localization of the membrane binding component of the complex, MinD, which in turn binds the inhibitor of FtsZ polymerization, MinC to prevent FtsZ polymerization at the poles [3]. Two modes of polar localization of MinD were found – static and dynamic. The static localization, like in Bacillus subtilis, supposedly involves another protein, DivIVA that localizes to the division site and consequently to poles, being attracted by division proteins or by the membrane curvature [4,5]. In Escherichia coli, MinD has a shorter membrane targeting sequence (MTS) that does not provide enough interaction energy to keep it on the membrane and thus requires at least dimerization of MinD to bind tightly [6]. An additional protein, MinE, impairs the MinD-membrane binding by inducing it to hydrolyze ATP, thus promoting deoligomerization [7,8]. This combination of MinD binding, MinE-induced detachment with diffusion along the cell and ADP-ATP exchange composes a highly dynamic, pole-to-pole oscillatory movement of this protein pair [9–12]. These features seem to be enough for the desired predominantly polar localization, supported by theoretical models [13–17].

At the same time, apparent preference of MinD for acidic phospholipids was demonstrated [6,18]. Together with the polar localization of cardiolipin (CL) both in B. subtilis [19] and in E. coli [20,21], these preferences could be an alternative or additional driving force for the MinD membrane distribution pattern. The distribution pattern of MinD appeared even more complex, with the oscillation wave moving along a helical path stretched from pole to pole [22,23]. Since no relationship was found with other membrane-associated helix-forming proteins like MreB [24] and considering that MinD binding to the membrane does not require any other protein, a pre-existing phospholipid heterogeneity of a particular pattern seems to be a reasonable possibility for the underlying binding structure. In addition to the above mentioned polar CL domains, lateral hetrogeneity of phospholipids in the cylindrical part of the cell was also evident from the uneven distribution of the fluorescent dye FM 4-64 [25]. Fluid membrane domains with a distinct order and polarity were detected and characterized in E. coli [26]. Dynamics of pyrene-labeled phospholipids, 2-pyrene-decanoyl-phosphatidylethanolamine (Py-PE) and 2-pyrene-decanoyl-phosphatidylglycerol (Py-PG) introduced into E. coli membrane indicated that the two phospholipids are sequestered into separate pre-existing domains in the bacterial membrane [27]. How could these phospholipid domains be formed and what could be their shape is not yet clear. Recently, a helical localization of integral membrane protein translocation complexes was demonstrated in B. subtilis [28] and E. coli [29]. Notably, the translocon components have distinctive preferences to acidic phospholipids for correct assembly and activity [30]. Whatever is the nature of the translocons’ helical localization, this structure should consequently be supplemented with the preferred acidic phospholipids. We hypothesize, that MinD follows this pattern in the course of its oscillations. The major phospholipids of the cytoplasmic E. coli membrane are phosphatidylethanolamine (PE, 70–75%), phosphatidylglycerol (PG, 20–25%), and CL (5–10%). Assuming that most of CL is located to poles, this PG content is enough for a reasonable binding of MinD [18]. However, it is not immediately clear that concentrating PG to a restricted area (e.g. helical domain) can produce a matching pattern of bound MinD. In support of this possibility, acidic phospholipid clusters, rather than randomly distributed PG, modulated the ligand binding affinity of peripheral DnaA [31] and phase separation of PG enhanced ATPase activity of integral SecA [32].

In the first part of this work [1] we investigated interaction between MinD and the membrane, focusing on the aspect of its influence on membrane structure and dynamics. Along with a general increase in the membrane order and viscosity, we observerd an apparent formation of acidic phospholipid domains around bound MinD at a particular mole ratio of PG in PC liposomes (see accompanying paper [1], Fig. 5). In this part, we aimed to examine whether binding of MinD to the membrane depends on lateral organization of acidic and zwitterionic phospholipids. For this, we have tested binding of MinD to two-component liposomes of various phospholipid compositions in which acidic phospholipids are either ideally mixed with zwitterionic, or concentrated to artificial domains as a result of phase separation. The phase separation was achieved in a binary mixture of phospholipids with very distant phase transition temperatures. Such mixtures display coexistence of gel and liquid-crystalline domains of individual phospholipids at their particular ratios and in specific temperature ranges [33]. We show here that binding of MinD is proportional to the PG fraction in the liquid membrane, but is strongly enhanced when PG is segregated into liquid domains in the surrounding gel-phase. Moreover, comparison of the phase transition profiles of mixed-phase liposomes in the absence and presence of MinD indicated that the protein affects the thermodynamic characteristics of the membrane that could be ascribed to stabilization of the PG liquid domain. These results strongly support the suggestion that concentrating acidic phospholipids into specific domains in bacterial membranes can shape the MinD distribution pattern in the cell.

Fig. 5.

Fig. 5

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Dependence of the membrane fluidity on the phospholipid head-group composition in SOPC liposomes with various fractions of SOPG, as shown. Membrane fluidity was manifested by anisotropy of DPH fluorescence (open squares), excimerization rate of pyrene (closed circles) or Py-PG (open circles). All values are normalized to those measured by each method in pure SOPG liposomes. Liposomes of desired composition were either labeled with 10−7 M DPH, or titrated with pyrene (see Methods in [1]). Py-PG was added to a set of phospholipid mixtures at 5 mole % fraction during liposome preparation.

Materials and Methods

Materials

1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC); 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG); 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC); 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) were purchased from Avanti Polar Lipids, Inc (Alabaster, AL). Chloroform was HPLC-grade. 1,6-diphenylhexatriene (DPH) and 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (Py-PG) were obtained from Molecular Probes (Eugene, OR). All other chemicals were analytical grade. Buffers were prepared in deionized water.

Expression and purification of MinD

His-MinD purification was performed exactly as described in the accompanying paper [1].

Preparation of Large Unilamellar Vesicles (LUV)

Phospholipids were dissolved in chloroform and dried to a thin layer film under a gentle steam of nitrogen. Dried phospholipids were hydrated in a 25 mM Tris-HCl, pH 7.5, 50 mM KCl buffer above the phase transition temperature for 1 h and then were vigorously vortexed. LUVs were prepared by extruding the suspension through a polycarbonate membrane filter (0.1 μm pore size, 19 mm diameter) 11 times using Avanti Mini-Extruder (Avanti Polar Lipids, Inc). The suspension and extruder were always kept above the phase transition temperature; LUVs were stored at −80°C. Phospholipids concentration was determined by phosphomolybdate colorimetric assay [34]. To prepare liposomes of desired composition, corresponding volumes of chloroform solutions of individual phospholipids were mixed and processed as described. The phospholipid composition is set as a mole percent of a particular kind and shown in % throughout the text, figures and legends.

MinD Binding to Liposomes

MinD stock solutions were centrifuged at 263,000 × g for 10 min at 4°C to remove potential aggregates. MinD (1.7 μM final concentration) was added to 65 μM LUV’s in 25 mM Tris-HCl, pH 7.5, 50 mM KCl, and 5 mM MgCl2 buffer with 1 mM ATP and incubated for 10 min at 37°C. After incubation, mixtures were centrifuged at 263,000 g at 37°C for 10 min. The pellets were resuspended in 40 μl sample buffer and subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with Coomassie Blue for 1 h and washed in 20% methanol–10% acetic acid overnight. Protein band densities were quantified by NIS Elements BR imaging software. A mixture without membranes, treated and analyzed likewise, was used as a control for protein sedimentation and a measure of remaining supernatant and was subtracted from all other samples.

Differential Scanning Calorimetry

Calorimetric experiments were performed on a MicroCal VP-DSC high-sensitivity differential scanning calorimeter (MicroCal, Inc., Northampton, MA, USA) applying a heating scan rate of 0.5°C/min. LUV’s were cycled five times through the phase transition temperature prior to measurements. The final lipid concentration in the DSC cuvette was 1 mg/ml. The on- (Ton) and offset (Toff) temperatures were determined by intersection of the peak slopes with the baseline of the thermograms. Ton and Toff were then corrected by the finite widths of the transitions of the pure components weighted with their mole fractions. The phase transition temperature (Tm) is taken as the temperature of maximum heat capacity after base line adjustment and normalization, using Microcal-Origin analytical software.

Fluorescence anisotropy temperature scans

Steady state fluorescence spectra and anisotropy were measured using Perkin-Elmer LS55 spectrofluorimeter (Perkin-Elmer, Beaconsfield, England) with a cuvette holder thermostated by a circulating water bath (F25ME, JULABO Labortechnik GmbH, Germany). The data were collected and analyzed with the dedicated software from Perkin-Elmer. DPH anisotropy in 50 μM LUV suspension and 0.5 μM DPH (1 mol % in the membrane, added as THF solution to suspension of membranes) was measured at 350 nm excitation and 450 nm emission wavelengths with 2.5/3 nm excitation and emission slits. Additional filters were placed in both the excitation and emission paths to reduce light scattering artifacts. Temperature scanning rate was controlled by the water bath at 1.33 °C/min for heating, and 0.6°C/min for cooling scans. The anisotropy readings were taken automatically with time intervals of 0.2 min, and the values were continuously acquired together with the readings of temperature in the cuvette holder. The temperature values were further corrected using calibration measurements in the cuvette. For measurements at a constant temperature, from 20 to 30 readings were taken after the sample equilibration and the average anisotropy value was obtained with a typical standard error less than 2%.

Measurements of the membrane dynamics with pyrene and Py-PG were performed as described in [1].

Results

1. Phase Transitions in two-component liposomes

For investigation of the presumed effects of phospholipid segregation into distinct domains on the MinD membrane binding, we exploited domains arising in liposomes made of mixtures of phospholipids with significantly different physico-chemical characteristics. One of the straightforward and evidently detected types of domains is the phase state one, in which phospholipid segregation is driven by a high difference in the individual phase transition temperatures. In the range between these temperatures, where one phospholipid is in the gel state and another in the liquid-crystalline, they are sorted out into domains of the corresponding phase state. Coexistence of such phase domains was shown in mixtures mimicking bacterial inner membrane composition [33] and were visualized in giant liposomes by fluorescence microscopy techniques [35,36]. Thermodynamic and order properties of liposome membranes composed of a variety of DSPC and SOPG mixtures (phase transition temperatures 55°C and −10°C, respectively) revealed by differential scanning calorimetry (DSC) and fluorescence anisotropy of DPH are presented below.

a. Phase transitions detected by DSC

Fig. 1 gives an overview of the DSC heating scans at several studied DSPC/SOPG ratios. The heat capacity peak sharpness and position for pure DSPC membrane are in a good agreement with the manufacturer’s data. Upon addition of SOPG to the mixture, broadening of the main phase transition peak and its shift toward lower temperatures were observed. The packing constraints for binary mixtures of DSPC and SOPG result from different molecular properties of the individual lipid components. SOPG phospholipids occupy a larger area derived from their geometrical size determined by both the head group and one unsaturated fatty acid and electrostatic repulsion between head groups. The pKapp of PG was determined to be 2.9 at physiological ionic strength [37] and it is therefore de-protonated in our experimental conditions compared with the uncharged DSPC with its relatively small head group and saturated fatty acid chains. The positions and shapes (width) of phase transitions were comparable in heating and cooling scans (not shown), adding to the reliability of the DSC data. Only the heating scans data were used to build the phase diagram.

Fig. 1.

Fig. 1

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DSC heating scans for DSPC/SOPG liposomes with different mole fractions of SOPG as shown. The heat capacity was measured as described in Materials and Methods and expressed in arbitrary units. The scan traces are shifted along the ordinate for a visual clarity.

b. Phase transitions detected by DPH fluorescence anisotropy

DPH was incorporated into the LUV bilayers of different phospholipids compositions as a fluorescent probe for the rotational diffusion of the surrounding phospholipid chains. The fluorescence anisotropy value (r) reflects the order-disorder transition in the membrane caused by temperature changes as shown in Fig. 2. DPH fluorescence anisotropy increases from the high values of about 0.25 representative for the ordered gel state to very low ones (~0.07) typical for a fluid membrane during the heating scans at all studied DSPC/SOPG ratios. Very similar shapes of cooling scans were obtained (data not shown). Again, the slopes of transition became less steep and shifted to lower temperatures with the increasing fraction of SOPG in the mixture.

Fig. 2.

Fig. 2

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DPH anisotropy heating scans for DSPC/SOPG liposomes with different mole fractions of SOPG as shown. DPH anisotropy was measured as described in Materials and Methods. The scans are shifted along the ordinate for a visual clarity; the interval between ticks is equal to 0.1 anisotropy units.

c. Phase Diagram

The results from the DSC (Fig. 1) and DPH anisotropy (Fig. 2) thermograms were used to build a phase diagram for DSPC/SOPG binary mixtures. In Fig. 3, the gel and liquid crystalline phase boundaries are constructed from the onset and offset temperatures of the phase transition data obtained for a variety of DSPC/SOPG ratios (see Methods and [33]). It is evident that a relatively broad fluid/gel coexistence region characterizes the DSPC/SOPG phase diagram. The membrane is apparently fluid and well mixed above this region and completely gel below. The transition temperatures obtained from DPH fluorescence anisotropy are in good agreement with determinations using DSC for LUV’s of same compositions. The best correspondence between the two methods is observed for 0–50% SOPG fractions and respectively high temperature transitions. Above 50% SOPG, the transition midpoint temperatures detected by DPH anisotropy are progressively lower than those measured by DSC. This discrepancy between thermodynamic and structural characteristics at increasing surface charge of the membrane may reflect the spatially different processes sensed by these methods. While DSC detects the general transition in the membrane, dependent on both electrostatic and hydrophobic interactions, DPH predominantly senses the fatty acid chains movement and packing. It appears that the order-disorder transition of the whole membrane requires more energy than the hydrophobic core alone when the fraction of charged phospholipids becomes dominant. The region of domain coexistence was further used to evaluate the effects of MinD attachment to a pre-existing phase domains.

Fig. 3.

Fig. 3

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Phase diagram presenting onset (circles) and offset (squares) temperatures of phase transitions detected by DSC (open symbols) and DPH anisotropy (closed symbols) scans as a function of a DSPC/SOPG composition. The area in-between the continuous lines connecting the corresponding on- and offset points relates to the gel-liquid-crystalline phase coexistence. The horizontal dashed line crosses the diagram at 37°C.

d. Phase states of liposomes of various compositions

Our aim was to compare MinD binding to the membrane containing a fraction of acidic phospholipids either well mixed with zwitterionic or concentrated to a domain. Using the described above temperature scans at particular SOPG fractions in the presence of MinD is disadvantageous because of i) too high temperatures needed to reach the liquid phase endangering the protein stability and ii) the necessity to take into account the temperature dependence of the binding itself and thus a difficulty to distinguish between impacts of different parameters. We therefore decided to examine MinD binding at a constant temperature but to membranes in which the phase state is determined by DSPC/SOPG ratio (marked by a dashed line in Fig. 3). This set is characterized by three parameters changing together with the SOPG fraction: surface charge, fatty acid saturation and, the desired, phase state. Accordingly, two additional phospholipid mixtures were used as controls: DSPG/SOPG and SOPC/SOPG. In the former mixture, thermotropic phase behavior similar to that of DSPC/SOPG is expected, since DSPG has the same phase transition temperature of 55°C as DSPC. At the same time, just two parameters –fatty acid saturation and phase state are variable at the permanent head group composition of DSPG/SOPG membranes. In the SOPC/SOPG mixture, only the surface charge is varied in the well-mixed membrane that will remain liquid over the whole range of ratios at 37°C.

To confirm that liposomes of chosen compositions indeed represent the whole range of the phase states at a fixed temperature, including the gel-liquid coexistence, fluorescence anisotropy of DPH in these liposomes was measured at 37°C. As shown in Fig. 4, high anisotropy values indicate the gel state of pure DSPC and DSPG vesicles (r~0.250), whereas only a value of r~0.075 characteristic for the liquid crystalline state was obtained at high fractions of SOPG with DSPG and DSPC. Notably, a phase transition-like behavior is displayed in the range of 30–70% of SOPG in DSPG/SOPG and DSPC/SOPG membranes, evident for the phase domain coexistence in these liposomes. The SOPC/SOPG membrane is liquid in the whole range of compositions and served as a homogeneous control for varying PG content. This variation of PG content affects also lipid mobility in a fluid membrane (Fig. 5).

Fig. 4.

Fig. 4

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Dependence of DPH anisotropy in SOPG/SOPC (closed squares), SOPG/DSPC (close circles), SOPG/DSPG (open circles) LUV’s on the fraction of SOPG at 37°C. The liposomes of various compositions were labeled with DPH and fluorescence anisotropy was measured as described in Materials and Methods.

Fig. 5 displays dependencies of the membrane fluidity on the head-group composition as reported by different probes, DPH, pyrene or Py-PG. These probes are reporting on different dynamic properties and from different localizations in the plane of the membrane (closer to the interface or in the hydrophobic core). While the order of the fatty acid core of the membrane sensed by DPH fluorescence anisotropy does not depend on the head-group nature of phospholipids possessing the same fatty acids (compare with Fig. 4), the lateral diffusion of pyrene and, even more, of Py-PG strongly increased with introduction of the negatively charged phospholipid into the zwitterionic membrane. This lipid lateral mobility, in addition to the membrane order, may be an important factor determining binding and activity of membrane proteins.

2. MinD binding is affected by pre-existing gel-liquid crystalline phases

MinD interactions with liposomes of various compositions of DSPC/SOPG, DSPG/SOPG, SOPC/SOPG phospholipids were analyzed at 37°C, a temperature at which the phase state of the membrane depends on phospholipid composition as described above. MinD-membrane interaction was monitored through the amount of protein that sediments with liposomes during ultracentrifugation (see Methods for details). MinD binding appears proportional to the SOPG fraction in the membrane composed of SOPC/SOPG (Fig. 6). These membranes remain in liquid crystalline phase at all ratios at the studied temperature (see Fig. 4) and thus this dependence may be ascribed mainly to the increasing electrostatic interaction between MinD and phospholipid head groups. In the liquid crystalline phase of all types of liposomes above 70% of SOPG fractions, it is notable that MinD has the association affinity with a preference order of DSPG>SOPC>DSPC as the second component. The highest attraction to the liquid DSPG/SOPG membrane containing only PG head groups looks obvious. MinD amphiphatic helix insertion into the DSPC-containing membrane is impeded presumably as a result of the two well packed saturated fatty acid chains, in contrast to the unsaturated SOPC (possessing the same head group) and in a good agreement with previous data on MinD binding preferences [18]. Protein binding to membranes that contain DSPC or DSPG show similar general dependence on the SOPG fraction, with a significant enhancement of binding in the range of phase coexistence (30–70% SOPG), when compared to membranes in a well mixed liquid-crystalline phase. The higher preference of MinD to such membranes is apparently due to SOPG segregation into a distinct liquid-crystalline phase domain in DSPC, and even DSPG, gel state environment. This enhancement disappears when the fraction of unsaturated SOPG is below 30%, the gel state boundary. It was also observed that for all membrane phases, MinD has the highest affinity to DSPG/SOPG membranes supporting its preference to anionic lipids.

Fig. 6.

Fig. 6

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MinD binding to DSPC/SOPG (squares), DSPG/SOPG (circles), SOPC/SOPG (triangles) mixtures at different fractions of SOPG. The binding was measured by co-sedimentation assay (see Material and Methods) and the data are normalized to the value for pure SOPG membrane in each set of liposomes. The phase states of DSPC/SOPG and DSPG/SOPG liposomes are roughly designated based on the data in Fig. 4.

3. MinD binding stabilizes the liquid SOPG domain

In the first part of this work [1] we have shown that binding of MinD to the membrane causes remarkable changes in its dynamic and structural properties, including the tendency to induce clustering of acidic phospholipids around the bound protein. All membranes, one- or two-component, used in these experiments were in the liquid-crystalline phase. It is expectable that MinD binding will also modify properties of the surrounding domain in a mixed-phase membrane. To investigate the influence of MinD binding on a model domain, liposomes composed of DSPC/SOPG at 52/48 molar ratio were used. According to the phase diagram (Fig. 3), these membranes have coexisting fluid and gel phases in the range of 30–45°C. We used heating temperature scans of DPH fluorescence anisotropy to detect fine changes in the phase transition characteristics of LUV’s, induced by MinD binding. It was found, as shown in Fig. 7, that the anisotropy thermorgrams for pure LUV’s or in the presence of MinD-ATP are similar but distinguishable. To get more detailed information on the transition characteristics, we fitted the experimental data with two sigmoidal functions, assuming a complex transition composed of a primary and secondary transition stages. Indeed, the two-sigmoidal fit gave better fitting quality (R=0.998) than the single one. Table 1 summarizes the temperatures and width of the two transition components for pure LUV’s and in the presence of MinD either in the ADP or ATP form. LUV phase transition is characterized by a narrow first phase at about 27.7°C and a broader primary transition at 39.7°C. No differences in transition characteristics were observed upon the addition of MinD-ADP to liposomes, according to its low binding capabilities. Addition of MinD-ATP caused an about 8°C up-shift of the secondary transition temperature with a pronounced peak broadening and only about 3°C shift of the narrowed primary transition peak. This up-shift of secondary transition temperature may be ascribed to a tightened packing of acidic phospholipids around the bound protein due to electrostatic interaction that increases the stability of the domain. Moreover, DPH anisotropy at low temperatures (gel phase) was higher in the presence of MinD-ATP, which reflects a more dense packing of the bilayer caused by protein binding. It should be mentioned, that analysis of the thermogram in the presence of MinD might be complicated due to the unknown temperature dependence of protein binding that may distort the shape of the thermogram. Moreover, the effect of protein may be diminished at temperatures higher than 45°C because of its denaturation.

Fig. 7.

Fig. 7

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DPH fluorescence anisotropy heating scan of DSPC/SOPG (52/48 molar ratio) LUV’s in the absence (dotted line) or presence (solid line) of 1.25 μM MinD. Fluorescence anisotropy (r) was measured in a vesicle suspension containing 50 μM lipid with 1 mM ATP and 1 mole % DPH as a function of temperature. The lines are the best fit with two sigmoidal functions (see text for explanations and the Table 1 for fitting parameters). Inset: first derivatives of the fitting function obtained with (solid line) and without (dotted line) MinD.

Table 1.

Comparison of the phase transition parameters in SOPG/DSPC membrane alone and in the presence of MinD-ATP or MinD-ADP as derived from two-sigmoidal fits to DPH anisotropy thermograms shown in Fig. 7.

Sample Anisotropy, r (below 10°C) Tsec (°C) Tsec1/2 (°C) Tpr (°C) Tpr1/2 (°C)
LUV’s (R=0.997, Chisq=0.011) 0.25±0.01 27.7±1.6 2.3±1.6 39.7±1.5 5.1±0.6
LUV’s-MinD-ADP (R= 0.997, Chisq=0.007) 0.26±0.03 25.1±0.4 0.5±0.4 39.4±0.3 4.5±0.2
LUV’s-MinD-ATP (R=0.998, Chisq=0.007) 0.28±0.01 35.7±0.8 6.1±0.2 42.9±0.6 1.8±0.8
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Tsec and Tpr – secondary and primary phase transition temperatures, Tsec1/2 and Tpr1/2 – their half-widths, correspondingly. The fit quality is shown in parentheses for each sample. Experimental conditions were as shown in the legend to Fig. 7.

Discussion

A model experimental system was established with the aim to examine whether binding of MinD to the membrane depends on lateral organization of acidic and zwitterionic phospholipids. For this purpose we have chosen segregation of two types of phospholipids into phase domains arising in consequence of their different thermotropic properties. Accordingly, the first stage of this work was to determine phase behavior of liposomes in wide ranges of phospholipid compositions and temperatures, commonly presented as phase diagrams. Three sets of binary mixtures were used: SOPG/SOPC, SOPG/DSPG and SOPG/DSPC. The first mixture is liquid at the temperatures tested and in the whole range of PG/PC ratios, while the last two exhibit phase coexistence (Fig. 3). Using this phase diagram we then chose a set of phospholipid compositions representing the whole array of phase states at a constant temperature of 37°C: coexistence of phases in the range of about 40–75% of PG and gel and liquid-crystalline phases below and above this range (Fig. 4).

Binding of MinD to liposomes of these compositions was tested by co-sedimentation method. The results presented in Fig. 6 clearly demonstrate the impact that the three membrane parameters - surface charge, fatty acid saturation and the phase state – have on the MinD binding affinity. The importance of the first two looks obvious in the view of the binding mechanism through amphiphilic MTS [6,38]. It involves initial electrostatic attraction to the membrane, followed by partial insertion enforced by hydrophobic interaction. Stronger negative surface charge and a looser packing of the membrane should encourage this binding. However, the binding is remarkably enhanced when the phospholipid carrying one these properties is concentrated to a domain. This is the case when SOPG is separated from the surrounding DSPC or DSPG. A simple explanation of this enhancement may be the possibility of sharing the boundary phospholipids by neighboring proteins, when these phospholipids are concentrated to a continuous field. Consequently, such a field is stabilized by the bound proteins (Fig. 7). In addition, the packing defects arising on the borders between phase domains may promote insertion of MTS into the membrane. However, their impact on the protein binding is undistinguishable from that of the liquid domain itself in experimental approach used in this work.

The type of membrane domains exploited in this work is an explicitly model one. By no means do we presume existence of phase domains in a native bacterial membrane at normal physiological conditions. Bacteria adjust their membrane composition to keep the liquid-crystalline state at different temperatures [39]. However, the order heterogeneity and segregation of phospholipids were shown to exist in a generally fluid bacterial membrane [26,27]. Our results suggest 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 modulating its temporal behavior.

Acknowledgments

A highly professional technical assistance of Sonia Soreanu is greatly appreciated. Dr. Sofia Kolushev is thanked for the aid with DSC measurements. This work was supported by grant nr. 820/05 from the Israel Science Foundation to IF, grants R37-GM20478 and R01-GM61074 from the United States National Institutes of General Medical Sciences to WD and WM, respectively.

Footnotes

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