Interaction of Polylysines with the Surface of Lipid Membranes

CHAPTER SIX

Interaction of Polylysines with the Surface of Lipid Membranes: The Electrostatic and Structural Aspects Natalia Marukovich*,†, Mark McMurray*,†, Olga Finogenova*, Alexey Nesterenko‡,}, Oleg Batishchev*, Yury Ermakov*,1

*Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia † Department of Molecular and Biological Physics, Moscow Institute of Physics and Technology, Moscow, Russia ‡ Division of Biophysics, Department of Biology, Lomonosov Moscow State University, Moscow, Russia } A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Materials and Methods 3. Electrokinetic Measurements in the Liposome Suspension 4. BP of Membranes 5. PL Adsorption at the Lipid Monolayers 6. Isothermal Titration Calorimetry of Liposomes in the Presence of PL 7. AFM of PLs at the Surface of Bilayer 8. Interaction of Lysine with Lipid Membranes 9. Lysine at the Membrane Surface: Analysis by MD 10. Speculations on PL Interaction with Lipid Bilayers Acknowledgments References

140 143 144 147 149 152 153 156 158 161 162 162

Abstract The topic correlates electrostatic effects induced by polylysine (PL) adsorption at the lipid membrane surface with data of alternative methods sensitive to lipid bilayer structure. Comparison of electrokinetic data for liposomes from anionic lipids (cardiolipin, phosphatidylserine) and results of boundary potential (BP) measurements with lipid membranes shows effects in two opposite directions: fast positive changes of BP due to adsorption of polycations at the outer membrane surface and slow negative changes that can be attributed to alteration of the dipole component of BP. The latter effect does not depend on the polymer length and may be caused by lipid interaction

Advances in Planar Lipid Bilayers and Liposomes, Volume 17 ISSN 1554-4516 http://dx.doi.org/10.1016/B978-0-12-411516-3.00006-1

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2013 Elsevier Inc. All rights reserved.

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with lysine as a basic unit of these polypeptides. Molecular dynamic simulation points out the possible mechanism of the dipole effect, which could be caused by reduced number of H-bonds to PO4 groups upon the lysine adsorption. Atomic force microscopy visualized the geometry of clusters formed by PL of different lengths at the lipid bilayer. Isotherm titration calorimetry and the technique of lipid monolayers reveal the similarity in polypeptide and inorganic multivalent cation effects on the lateral lipid condensation accompanied by dipole effects.

1. INTRODUCTION Lipid membranes are well-known models of cell membranes and are commonly used to study their interactions with biologically active substances. Adsorption of polypeptides modifies the physical state of the lipid matrix of cell membranes; that is why it has to be thoroughly studied before application of these polypeptides in medicine and biotechnology. Particularly, the electrostatic interaction of polylysines (PLs) with negatively charged membranes is the typical object of research in biochemistry and colloid chemistry, stimulated by thorough development of its biomedical applications [1,2]. Research clearly shows that PL adsorption depends on the presence of anionic lipids at the membrane outer surface [3–6]. Lipid systems—liposomes, planar bilayer lipid membranes (BLM) or lipid monolayers—composed of anionic phospholipids cardiolipin (CL) and phosphatidylserine (PS) have a special significance in cell biology: CL is a specific lipid of mitochondria participating in energy production [7,8]; PS is widely distributed in different tissues and takes part in Ca regulation, membrane targeting, etc. [9]; both lipids are seen as key participants in apoptosis [9,10]. These lipids impart the negative charge to cell membranes and act as the binding site for inorganic cations and positively charged macromolecules such as polypeptides. The artificial lipid systems are well suited for studying electrostatic interactions of PL with cell membranes; therefore they are actively used in polymer science and membrane biophysics for the development of fundamental theories [1,11–13] and experimental approaches [4,14–19]. Certainly, we do not aim here to give a comprehensive review of the current state of these studies. We direct readers to the papers cited in the chapter for a deep understanding this wide field of research. Most of them investigate the structure of polymer layers, conformation of macromolecules, and their ability to create significant defects in biomembranes, controlling their conductivity as a direct way to affect

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membrane biochemistry. On the other hand, polypeptide interaction with the lipid matrix of biomembranes may control the conformational lability of membrane proteins by changing the elasticity of their environment as it was recently revealed for the blocking effect of multivalent cations on mechanosensitive channels [20]. It was found that the dipole potential at the membrane boundaries indicates the related lateral condensation of lipids [21]. Nevertheless, the knowledge of these aspects of polymer–lipid interactions is incomplete both in the experimental approach and in the qualitative descriptions of general phenomena. These facts called our attention to the electrostatic and thermodynamic phenomena accompanied by adsorption of polypeptides. They provided a way to find the similarity and the specificity of PL, single molecules of lysine, and multivalent cations in their influence on the lipid packing as it follows from the electric field distribution over the membrane/water interface and from complimentary methods. Generally, lipid packing and polymer conformation at the membrane surface depend on the peptide composition, chain length, and conformation as it follows from data of differential scanning and titration calorimetry [22,23], FT-IR and Raman spectroscopy [24,25], and NMR [26]. On the other hand, the adsorption of PL and other positively charged macromolecules induce domain formation in the lipid bilayer enriched with a negatively charged component [6,24,27–31]. Actually, these phenomena are accompanied by significant changes in the electric field distribution at the membrane surface, so electrostatic measurements become very informative. We used them together with other complementary methods to detect specific changes that can be referred to as the alteration of the membrane structure. In particular, electrostatic effects induced by PL adsorption at the membrane surface were registered by the traditional electrokinetic method in liposome suspension and by the method of intramembranous field compensation (IFC) developed for planar BLM [32]. The latter technique has some advantages in visualizing the complicated kinetics accompanied by PL adsorption and report on the intrinsic events in the lipid bilayer. In our experience, the changes in the dipole component of total potential drop at the membrane boundary (boundary potential, BP) induced by inorganic ions of high affinity to lipids indicate an alteration of lipid packing and their physical state [20,21]. Application of the same approach to more complicated polymer–lipid systems assumes a correlation of electrostatic phenomena with alternative methods sensitive to polymer–lipid interaction, for example, titration calorimetry, monolayer technique, and atomic force microscopy (AFM). Here, we present some critical experiments carried

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out by the above-mentioned methods confirming PL effect on the membrane structure. Simulations of the system by molecular dynamics (MD) provided some assumptions about details of PL–lipid interaction determined by lysine–lipid contacts. We realize that our current data are not fully completed for quantitative analysis in the framework of any theoretical model, as they are very preliminary. In any case, we have decided to share our observations, which reveal some unexpected facts and open new important features of PL–lipid complexes. The electric field at the membrane boundaries, BP, consists of two principal components: the electric potential drop in the diffuse part of the electrical double layer (EDL) here called the surface potential and the potential drop in the polar region of the lipid bilayer. The greatest contribution to the latter component of BP comes from the orientation of the intrinsic dipole moments of lipids and, to a greater extent, from associated water molecules. So, we refer to this component as the dipole potential even though it may have another nature in some specific cases [33]. The treatment of corresponding data by the Gouy–Chapman–Stern model seems to be their more adequate description in real accuracy of the measurements [34]. This approach was developed in Ref. [35] and described in detail by many reviews and monographs, cited in Ref. [36]. But the second part of BP is much more interesting if the structure of lipid bilayer is expected to alter in the presence of biologically important ions and substances. It is important to note that not the absolute value of the dipole potential but its deviations can be measured directly in contrast to surface potential [37]. One possibility is to use hydrophobic ions because their penetration through the lipid bilayer is sensitive to total potential drop over the membrane boundaries [35,36]. But this method has the general disadvantages typical for any membrane probes: the corresponding data strongly depend on the nature of organic ions and their mobility in the hydrophobic region of the membrane [38]. An alternative way to measure the changes of BP dipole component was developed in our laboratory and applied to study electrostatic effects induced in planar BLM by different membrane active substances. Its principles are described in a review [32] and original papers [21,33]. In short, the electric capacitance of BLM is “elastic” and because of this its magnitude is sensitive to the electric potential inside the hydrophobic region of the membrane. If no external voltage is applied to BLM, the potential drop over the hydrophobic part of the lipid bilayer reflects the electrostatic asymmetry between membrane sides. It is typically caused by any charged substances adsorbed at one BLM side (cis-side) if they cannot penetrate to the opposite (trans) side

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that can be used as a reference. These substances, inorganic ions, small molecules, or ionized polyelectrolytes may generally affect the total BP by changes in potential distribution near the membrane surface and/or in the region of lipid polar heads. The magnitude of the dipole component of BP may indicate these changes. The difference of BP appearing between cis and trans BLM sides can be compensated, and IFC technique records this difference in time. So, it allows one to control the electrostatic phenomena accompanied by adsorption of substances at BLM surface and to test their reversibility by permanent perfusion of the cell. This facility of the IFC method appears important in our studies.

2. MATERIALS AND METHODS Most experiments presented and discussed below were performed on a self-made setup of IFC technique with the planar BLM described in Ref. [32] combined with the traditional electrokinetic measurements using dynamic light scattering technique (Zetasiser-2 Malvern Instr., UK). The typical experiments were carried out with lipid systems composed of lipids negatively charged at normal pH (CL or PS) and their mixture with neutral phosphatidylcholine (PC) (“Sigma” or Avanti Polar lipids) in monovalent electrolyte (10 mM KCl if not shown otherwise) at room temperature. Samples of PLs of different molecular masses (“Sigma”) are listed in Table 6.1 together with their commercial parameters. The other experimental details are listed in the text. Table 6.1 Lysine and PL products Notation and number of units Name

Molecular weight Catalog by viscosity number sigma

Lysine

L-Lysine, D-lysine Monohydrochloride

182.65

L-8662

PL-5

Pentalysine

658.9

L-9151

PL-12

Poly-D-lysine hydrobromide 2500

P-0296

PL-130

Poly-D-lysine hydrobromide 27,200

P-4408

PL-211

Poly-D-lysine hydrobromide 44,100

P-7886

PL-235

Poly-D-lysine hydrobromide 53,000

P-7886

PL-598

Poly-D-lysine hydrobromide 125,100

P-0899

PL-1435

Poly-D-lysine hydrobromide 300,000

P-1149

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Experiments with lipid monolayers were done with dimyristoylphosphatidylserine (DMPS) on device NIMA Type 601 (25
20 cm) Langmuir Film Balance. Isotherm titration calorimetry (ITC) measurements were conducted on a Microcal VP-ITC calorimeter. Prior to injections, the cell was equilibrated at either 25 or 45  C to perform measurements on lipids below or above the temperature of phase transition (around 40  C for DMPS in KCl electrolytes). The heat responses were integrated and analyzed with the MicroCal Origin (version 7) software. Data of AFM were obtained with multimode microscope (Bruker, USA) equipped with Nanoscope IV controller, E-type scanning head, and a cell for electrochemical measurements in liquids. Experiments were carried out at room temperature in the tapping mode in background solution (10 mM KCl). The probes (NP-type cantilevers) of silicon nitride have a nominal spring constant of 0.12 N/m (Veeco Instruments Inc., USA). The diameter of the cantilever tip was 20 nm. Formation of supported lipid bilayers on freshly cleaved mica was performed by deposing the liposomes (1 mg/ml, sonicated liposomes) of corresponding lipid composition on freshly cleaved mica (200 ml/2.5 cm2) followed by incubation for 1 h at room temperature. Then, the sample was rinsed five times with background solution. MD simulations were performed using all-atom force field OPLS-AA with explicit water TIP4P on GROMACS [39] with periodic boundary conditions and a time step of 0.5 fs. Dioleoyl phosphatidylserine (DOPS) bilayers were simulated in NpT ensemble at 300 K. Membrane was constructed from hexagonally packed lipids and equilibrated in KCl solution for about 100 ns, then the Kþ/lysineþ ratio was varied and the system was equilibrated again for 40 ns. A subsequent 60 ns of dynamics were taken for final calculations. Lipid topologies, MD protocols, and data preparation features are described in detail in Refs. [40,41]. All of these methods are well described in the literature cited below, which is why we present here our data with no detailed explanation of the technical details. The principal conditions and the measurement procedure are described at the proper places of the text.

3. ELECTROKINETIC MEASUREMENTS IN THE LIPOSOME SUSPENSION Electrokinetic data are known in the literature for polyelectrolytes of different chemical nature: natural peptides, synthetic polymers, and polypeptides [42–45]. These macromolecules are polycations and show similar

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electrostatic effects detected by electrokinetic measurements. Mobility of negatively charged liposomes in these experiments demonstrates a sharp dependence on polyelectrolyte amount in the suspension, which corresponds to a very narrow range of concentration (in bases) and overcharge effect at the saturation level of mobility [46–48]. We have found the same results with PLs of different molecular weights adsorbed at liposomes composed from CL, PS, or their mixture with PC [46]. No effect was found with neutral liposomes from pure PC. Several curves in Fig. 6.1 demonstrate a difference between lysine, its oligomers, and PLs of high-molecular weight in their effect on the electrophoretic mobility of multilayer liposomes made from CL. Lysine adsorption slightly shifts the surface charge of liposomes in a positive direction, small polypeptides neutralize liposomes, and PLs with long chain saturate and overcharge the surface charge. The saturation level has the same magnitude for all PL macromolecules. Note that the experimental data are presented in the scale of zeta-potential, z, calculated from measured mobility values, m, by Smoluchowski relation, z ¼ m
/ee0, where
is viscosity and ee0 are usual dielectric parameters of the water media. In fact, this treatment is not correct because it assumes an ideal smooth surface for electrostatics and hydrodynamics PL-598

100

PL-211 PL-130 PL-75

Zeta potential (mV)

PL-12

50

PL-12 PL-5 PL-4 Lysine

0

−50

−100 −6

−5

−4 −3 Lg[C] (Munits)

−2

−1

Figure 6.1 Zeta-potential of CL liposomes measured at stepwise increase of the concentration of polylysines in background electrolyte 10 mM KCl, pH about 7.0. The data for PL-5, PL-12, PL-139, and PL-211 are from Ref. [46]. Amount of lipid in the suspension is 1 mg/ml.

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of polymer surface exposed to water. A more realistic situation was suggested in the theoretical approach developed in Refs. [49–51]. Unfortunately, the complicated theories introduce a lot of fitting parameters that are not available for direct experimental control. That is why we prefer to use the simplest models of EDL to evaluate semiquantitative parameters—surface potential and charge density—to follow their dependence on system conditions. The saturation level of surface charge density of PL layer seems to be in linear dependence on the charge density of intact liposomes, but these charges have an opposite sign. The positive values of surface charge density of PL layer are about twice the negative ones of intact liposomes. According to electrokinetic data, these values depend on the membrane surface charge and qualitatively correspond to the traditional Gouy–Chapman model of EDL [46,52]. If no polymer layer exists at the liposome surface, this model fits well with the experimental data with the assumption that zeta-potentials are measured in shearing plane at some distance from the plane of charges. This distance was evaluated and defined as being equal to 0.2 nm by comparison of electrokinetic data with alternative methods sensitive to electric field distribution over membrane boundaries, by measurement of hydrophobic ion mobility inside the membrane combined with fluorescent probes [35,53], or by the method of IFC [32,33]. In the case of PL layer at the liposome surface, these definitions are invalid and the surface charge density was evaluated as 0.005 and 0.016 C/m2 for polymers with 5 and 12 units (lysines), respectively, and 0.032 C/m2 for PL with 130 and 1435 units with the assumption that shearing plane coincides with the surface [52]. The narrow range of PL concentration around zero charge point, shown in Fig. 6.1, is qualitatively similar to what we observed in the case of multivalent cations Gd3þ and Be2þ [21]. Obviously, the electrostatic effects with inorganic ions and polycations have a different physical nature. Inorganic ions are in normal equilibrium between the bulk and membrane surface, and experimental data can be formally described by Langmuir type of isotherm with extremely large binding constants (about 50,000 per M). Undoubtedly, this interpretation is invalid for polycation irreversible adsorption. It is important to note that the binding of multivalent cations includes a process of lipid condensation observed by the technique of Langmuir monolayers and detected by ITC [20]. The specificity of polymer with a long chain looks natural: the charged units facilitate the cooperative binding to negatively charged sites of neighboring units. This idea was tested in Refs. [12,54] with lysine oligomers in PS–PC mixed systems. On the other hand, desorption of polycations became impossible because all contacts must

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75

CL (mg/ml) 0.2

Zeta potential (mV)

50

2.0 1.0

25 0

−25

−6.0

−5.0

−4.0

−3.0 Lg[C] (Munits)

−50 −75 −100

Figure 6.2 Zeta-potential of CL liposome measured at stepwise increase of the concentration of polylysine PL-598 in background electrolyte 10 mM KCl, pH about 7.0 with varied amount of lipid shown in the picture.

be broken simultaneously. This means that there is no equilibrium between polymer molecules at the surface and in the bulk, and no free macromolecules exist in the suspension before the total surface of the membranes is occupied by polymer. This fact was proved directly with PLs in Ref. [48], and tested in our experiments. Particularly, experimental curves in Fig. 6.2 correspond to various amounts of lipid in liposome suspension. This amount is roughly proportional to the total area of the liposome surface exposed to the solution. All experiments show the same saturation level, but the position of zero charge point is shifted to smaller PL concentration with decreased lipid content in the suspension. The only explanation of this fact is irreversible adsorption of polymer. Another argument that supported this conclusion was found by perfusion of the cell with planar BLM controlled by IFC method. No changes of BLM BP were registered when adsorbed PLs saturated the membrane surface and then washed out of the cell by background electrolyte. Moreover, kinetics of potential changes at the intermediate levels of PL adsorption is not connected to polypeptide removal from the cell, as it follows from the data presented in the next section.

4. BP OF MEMBRANES The dipole component of BP is generated by dipoles of lipid head groups and associated water molecules, and thus, it reflects the lipid packing and structure reorganization induced by adsorbed substances [55–58]. This

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component cannot be measured directly but its deviations may be controlled indirectly by different dipole sensitive probes [55,57,59,60] or by subtracting electric field changes in the diffuse part of EDL (zeta-potentials) from total BP that comes, for instance, from direct Volta potential measurements at lipid monolayer [37,55] or from IFC measurements with planar BLM. The latter method, described in detail in Ref. [32], assumes that the planar BLM conductivity remains negligible in the presence of adsorbed polymer and this condition has to be carefully controlled because PL of high-molecular weight decrease the BLM stability to applied external voltage [14]. Fortunately, these factors have no influence on IFC data in our experimental conditions. The IFC method applied to planar BLM detects changes of BP at one membrane side (cis-side) keeping the other (trans-side) as a reference. Moreover, it allows controlling the kinetics of adsorption and desorption processes in response to added substances to the proper cell compartment (about 1 ml) or to perfusion of cis-compartment by background electrolyte. The typical records of both processes are shown in Fig. 6.3 for the case of lysine adsorption. Arrows in the lower part of the figure show moments for lysine stock solution added to cis-side of BLM with intensive stirring of the solution in the cell. BP shifts stepwise in a positive direction within a short time due to lysine adsorption. The time constant of lysine removal from the

Boundary potencial (mV)

50 40 30 20 10 0 0

10

20 30 Time (min)

40

50

Figure 6.3 Boundary potential measured by IFC method with planar BLM from CL under stepwise increase of lysine concentration in the cell from 1 to 9.7 mM at the moments shown by arrows under the curve, and during lysine washing out by permanent perfusion of the cell (2 ml) with background electrolyte (10 mM KCl) at flow 0.3 ml/min. Thick arrows at the top show on and off moments of the pump.

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cell is about 10 min and depends on flow velocity of the background electrolyte through the cis-compartment of the cell (about 1 ml/min). This experiment clearly demonstrates the reversibility of lysine binding to the membrane, such as observed for inorganic ions and many small molecules. That is not the case for polypeptides of any molecular weight. The experimental record in Fig. 6.4A demonstrates the same experiment with pentalysine. It was added to the cell compartment on one BLM side (lowest arrows) and then removed from the cell by perfusion (noisy sections of the curve correspond to a working pump). At the first pump switch, the BP decreases as for lysine in Fig. 6.3. Initially, we were confused by the interpretation of this step and attributed it to the process of pentalysine desorption [46]. But now, we have found that fast changes of BP in a positive direction are followed by negative changes independently if the pump is on or off (see second section of the curve Fig. 6.4A). The same set of experiments with similar observations were done with PL of different chain lengths (Fig. 6.4B). These experiments reveal the changes of BP in two phases in opposite directions. The second negative phase has almost the same time (about 10–20 min) and magnitude (around 40 mV) and appears at the intermediate states of surface saturation by any studied PLs. Without doubt, large charged macromolecules cannot penetrate the membrane and so affect the BP at the opposite BLM side. So, we may conclude that the fast positive shift of BP corresponds to polycation adsorption at the membrane surface and the negative phase seems to report on alterations of bilayer structure that are indicated by negative changes of dipole potential. It is necessary to note that in the case of multivalent inorganic cations the dipole potential becomes more positive by up to 150 mV [21].

5. PL ADSORPTION AT THE LIPID MONOLAYERS The condensation of lipids in monolayers was registered in the presence of PL such as it was found with multivalent inorganic cations in Ref. [20]. Area-pressure isotherms of DMPS monolayers presented in Fig. 6.5 were measured in the presence of lysine, pentalysine, and PL in subphase. To compare the shape of the experimental curves, they were shifted in abscissa axis to the same position of the steepest parts. These isotherms clearly demonstrate that the pressure decreases in the presence of PL and lysine and the slope of all curves increases in the region of liquid-expanded state of monolayers. This fact is a qualitative sign of decreased compressibility of monolayer (increased rigidity). Almost the same result was found with multivalent cations, but the

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A

80 70

Boundary potential (mV)

60 50 40 30 20 10 0 −10 0

50

100

150 200 Time (min)

250

300

350

B

Boundary potential (mV)

150

100

50

0 0

50

100 150 Time (min)

200

250

Figure 6.4 (A) Experimental record of boundary potential changes due to PL-5 added at cis-side of BLM in the moments shown by arrows under the curve and washed out by permanent perfusion. Thick arrows correspond to on and off moments of the pump. (B) Typical record of boundary potential changes due to polylysine added stepwise at cis-side of BLM at the moments shown under the curve. Concentration of PL-12 in the cell was increased from 5 to 50 mM.

effect was much more pronounced. In contrast to PL, in the case of Gd3þ liquid-expanded state of monolayer disappeared and monolayer demonstrated the rigidity of gel phase in the whole range of areas per lipid. Lysine and pentalysine added to subphase is immediately reflected by the compression of monolayer, but in the case of PL it takes some time. The isotherm shown in this figure for PL was measured in three steps: monolayer was

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Pressure (mN/m)

mN/m 60

10

50

5

40

0

5

0

10 min

30 a

20 b

c

d

10 0

20

40

60 80 Area per mol (A2)

100

120

Figure 6.5 Pressure-area diagrams of DMPS monolayers measured with a different composition of background electrolyte in subphase: a, control 10 mM KCl; b, 2 mM lysine in 10 mM KCl; c, 0.075 mg/ml of PL-5 in 10 mM KCl; d, 0.048 mM of PL-235 in 100 mM KCl, 10 mM Tris. Total amount of the lipid in the bath was 2.25
104 mg/ml. The inset is the pressure decreased at fixed surface area in the experiment with PL-235 (d).

initially compressed by a barrier up to a pressure of about 14 dyn/cm, then the barrier was stopped and the decay of the pressure was recorded in time (inset in the figure), and finally, the compression was continued up to destruction of the monolayer. The slope of the curves at the latter step indicates a compressibility similar for pure DMPS monolayer and in the presence of all studied substances. The time-dependent decrease in pressure at the second step looks similar to the kinetics of BP changes in IFC experiments (Fig. 6.4). Generally, domain formation is assumed as a molecular mechanism of liquid-gel phase transition, and inorganic cations facilitate this process and make the monolayer more rigid at any area per lipid [20]. One may expect the same phenomena also in the case of PL. This process may be limited in time by diffusion of polymer through the unstirred layer at the surface and/or by formation of cluster-like structures in the lipid monolayer. Interpretation of these kinetics is more complicated because it may be accompanied by folding of the polypeptide chain and other transformations induced by phase transition in lipid monolayer [61]. But it is important to note that the “shoulder” of liquid-gel phase transition remains at the same position of the area per lipid for all diagrams. So, PL has a small effect on this transition and compression of monolayer may have other effects in the case of multivalent cations.

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6. ISOTHERMAL TITRATION CALORIMETRY OF LIPOSOMES IN THE PRESENCE OF PL We have compared multivalent cations and PL adsorption at the membranes with lipids in solid and fluid states. We did it by means of titration calorimetry with liposomes from DMPS. Two series of cumulative heat (Fig. 6.6) were measured by stepwise PL injection to DMPS liposomes at temperatures fixed lower and higher than that of DMPS main phase transition (around 40  C). The results are quite similar to that for Gd3þ cations [20]. When the membranes are in a fluid state, the adsorption of these cations and PL is an exothermic process similar to that published by other authors [23]. Significantly, we have different results for liposomes in solid (gel) state—this process became endothermic. In both cases, we observed a specific point in the scale of solute–lipid ratio that quantitatively corresponded to zero charge point according to our electrokinetic data. So, the physical mechanism of cation–surface interaction is dramatically changed at the surface neutralization. The increment of free energy due to surface charge

1.0

Solid (25 ⬚C)

0.8

Liquid (45 ⬚C)

kcal/mole of injectant

0.6 0.4 0.2 0.0 −0.2 −0.4 −0.6 −0.8 −1.0 0.00

0.05

0.10

0.15

0.20

0.25

0.30

Molar ratio

Figure 6.6 Cumulative heat measured by PL-235 injections to suspension of DMPS liposomes in 10 mM KCl at temperatures fixed lower and higher than the temperature of liquid-gel phase transition. The ratio of polymer–lipid around 0.18 corresponds to zero charge point.

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neutralization can be evaluated by well-known equations of the Gouy–Chapman model (see e.g., Ref. [36]). It does not exceed 1 mcal/M of charge if intact liposomes have about 100 mV surface potential in 10 mM monovalent electrolyte. This value is comparable to the magnitude of enthalpy changes observed in the exothermic branch of the experimental curve in Fig. 6.6. So, we may attribute it to electrostatic phenomena only. That is why we hesitate to use here the theoretical approach developed in Ref. [23] and typical software suggested by Microcal Company. Note that in the case of multivalent cations, the changes of enthalpy are several times greater and include processes such as phase transition. PL adsorption at the surface of “solid” liposomes is endothermic and is accompanied by smaller changes of enthalpy. Unfortunately, we did not find an adequate theory to describe this phenomenon. It seems a realistic assumption that it is of an entropic nature and related to water redistribution at the membrane interface between lipid and polymer. This idea encourages us to develop MD simulation of this system in the near future.

7. AFM OF PLs AT THE SURFACE OF BILAYER Irreversible adsorption is the important property of PL used in many technical applications and for studies of the membrane surface by AFM. We used this method to evaluate the size of polymer clusters at the surface of lipid bilayers of varied composition. Lipid bilayers were formed at mica surface from suspension of liposomes made with a mixture of CL/PC. Their bilayer structure was tested by force distance curves [62,63]. Then the bilayer was exposed to PL solution for about 1 h for surface saturation, and unbound polymer was removed from the cell (but not from the surface!) by rinsing with background electrolyte (10 mM KCl). Data of quantitative analysis of scanned surface in the presence of different PLs are presented in Table 6.2 and illustrated in Fig. 6.7. Table 6.2 Data of AFM images: The relative occupied area, A and the average thickness of polymer layer, D CL in the mixture No polymer PL-5 PL-130 PL-1435

20%

A (%) D (nm)

27  3 0.5  0.2

30  3 1.5  0.4

37  3 1.7  0.5

45  1 2.5  0.5

40%

A (%) D (nm)

39  2 0.6  0.2

39  3 1.5  0.4

Unresolved

Unresolved

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2,0

A

20% CL

C

1,5

1,0

D

.7 nm

0,5

Z: 2

0,0 0

250

500

750

1000

750

1000

750

1000

750

1000

X (nm) 3

m

.0 m

X: 1

D

2

D 1

0 0

250

500

X (nm) Z (nm)

B 20% CL + PL-5

3

E

2

.1 nm

D

Z: 5

1

0 250

0

500

X (nm) 6

.0 mm

F

Z (nm)

X: 1

4

D

2

0 0

250

500

X (nm)

Figure 6.7 AFM data on PL at the supported bilayers: 3D images of scanned bilayer with 20% CL (A) and adsorbed PL-5 (B). Height profiles for 20% CL bilayer (C) and adsorbed PL-5 (D), PL-130 (E), and PL-1435 (F). Horizontal lines and shaded areas are shown to discriminate the pikes from the background. The average thickness of polymer layers, D, is listed in Table 6.2.

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The 3D examples of scanned topography images, shown in Fig. 6.7A and B, correspond to lipid bilayers composed of 20% CL in the CL/PC mixture and that with adsorbed PL-5. Several cross sections of height profile for the topography image are shown in Fig. 6.7C–F with shaded areas that discriminate pikes from the background. All scanned images have two types of objects: bright peaks and dark gray background. To evaluate the area occupied by higher objects, the height distribution diagrams for the images were plotted by WSxM software [64]. The height distribution histograms were fitted by combination of two Gaussian curves. The area under the curve corresponding to the bright regions normalized to the whole area of the image was used to estimate the area occupied by higher objects in the scanned image. These data are listed in Table 6.2. In the case of free bilayer, the higher peaks (Fig. 6.7A and C) refer to CL molecules and the other part of the surface to PC. The visible protrusion of CL molecules from the lipid bilayer is possibly caused by repulsion of the negatively charged SiN3 cantilever [65] from ionized CL molecules. As it follows from some studies [66], the electrostatic interaction between the tip and the sample should be taken into account at low ionic strengths. It means that if the tip and some object in the sample have the same sign of charge the apparent height of this object increases. In accordance with this consideration, the total cross-sectional area occupied by bright objects in Fig. 6.7A shows an area well correlated with the percentage of CL in the mixture (Table 6.2). That is, CL molecules in the supported bilayer are initially organized in charged domains. A similar phenomenon was described in recent studies of more complicated domain structure in bilayers of mixed compositions [16]. Our experiments show that PLs adsorbed at the supported bilayer do not induce lipid segregation from the uniform surface, but they cover the negatively charged domains. In the case of PL-5, the domain distribution and the total area of clusters with this oligomer are quite similar to the initial domain distribution in spite of the height difference between baseline and maximal peaks (Fig. 6.7D). The difference between heights of free and occupied areas is small and the oligomer layer seems to be flat. Adsorption of PL-5 at negatively charged CL domains does not change their size (or composition). That is not the case for PLs of high-molecular weight. According to our data, PL-130 and PL-1435 make clusters with a total area about three times more than the initial domain distribution (Table 6.2); hence, polymers with long chains cover some parts of the uncharged surface of the bilayer. It is interesting to note that the thickness of the layer with PL-130 is approximately equal to one for PL-5 oligomer (Fig. 6.7E). The layer with long PL-1435 is much

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thicker, up to 2.4 nm, but with the same surface area as with PL-130 (Fig. 6.7F). That is, the polymer conformation is different in both cases, and it is possible that the longest PL have coils near the surface. In accordance with Ref. [61], we may assume that these conformations are related to beta and alpha structures of PL-130 and PL-1435, respectively.

8. INTERACTION OF LYSINE WITH LIPID MEMBRANES Experimental data presented here demonstrate a serious alteration in lipid membranes induced by PL adsorption at their surface. This was indicated by changes in the dipole component of BP, the compressibility of lipid monolayers, and from the packing of lipids in domains stimulated by polypeptides. The physical basis of these phenomena is a specific property of lipid polar heads to be ionized and to contact charged groups of polypeptide. The hydration effect seems to be most important in this respect. This conclusion is well supported by our experiments in the simple example of lysine adsorption. Two series of measurements are compared in Fig. 6.8 and clearly reveal a difference between surface and BPs of the membranes from PS in their dependence on lysine concentration. As seen from the kinetic experiments shown in Fig. 6.3, lysine adsorption is totally reversible. Because of equilibrium between lysine at the surface and in the bulk, we can use a normal approach to theoretical calculations of the surface potential. The traditional theory of EDL and Gouy–Chapman–Stern equations are well suited for this purpose. Theoretical curves in Fig. 6.8 correspond to Langmuir-type isotherm for two monovalent cations, lysine and potassium, competitively bound to negatively charged binding sites, assumed as a single ionized PS molecule. s s max

¼

1 : 1 þ K1 c1 ð0Þ þ K2 c2 ð0Þ

Here, ci(0) is the concentration of each cation at the membrane surface, that is, distributed in the diffuse layer of the EDL according to Boltzmann relation: ci ð0Þ ¼ ci,bulk exp

ezi fðxÞ : kT

The surface potential, f, and surface charge density, s, are related by the formula of Grahame [67], which takes into account all cations and anions in the electrolyte of arbitrary composition:

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0.04

100 80

0

60

s

40 20 0 −4

−3

−2

−1

0

Surface charge (C/m2)

Surface potential (mV)

f

−0.06

Lg[C] (M)

Figure 6.8 The increment of zeta-potential of liposomes (open points) and boundary potential of planar BLM (crosses) measured in 10 mM KCl and membranes from PS by electrokinetic and IFC methods, respectively, at different lysine concentration. Solid and dashed curves are calculated for surface potential (f, left ordinate) and surface charge density (s, right ordinate) according to the Gouy–Chapmen–Stern model of the electrical double layer, taking into account the competitive adsorption of potassium cations and lysine with binding constants 0.2 and 1.1 per M, respectively.

s20

¼ 2kT ee0

X

 ci,bulk

i

   ezi ’ð0Þ exp  1 : kT

Naturally, these equations simplified the system—any ions and lysine molecules are assumed as “nondimensional” points with a similar contribution to electrolyte ionic strength, I, and Debye screening lengths, L: X I ¼ 0:5 ci,bulk z2i , 

i 2

2e ci,bulk 1 ¼ ee0 kT L

1=2 :

And finally, the shearing plane for zeta-potential electrokinetic measurements is assumed at a distance of d ¼ 0.2 nm from the ideal smooth surface. It is remarkable that the simplest theory describes well the electrokinetic data by three fitting parameters—maximal surface charge density, smax ¼ 0.12 C/m2 and binding constants K1 ¼ 0.2 per M for potassium ions with fixed bulk concentration ci,bulk ¼ 0.01 M and K2 ¼ 1.1 per M for lysine with varied concentration as a monovalent cation. This theoretical approximation predicts the asymptote for surface potential to be about 58 mV per decade of monovalent electrolyte concentration and small variation of surface charge density (dashed line in Fig. 6.8). Note that the asymptote has the same slope in the experiments

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made by both methods. It is important that the occupancy of the surface by potassium and lysine evaluated by the theory of EDL can be used for comparison with the digital experiment by means of MD.

9. LYSINE AT THE MEMBRANE SURFACE: ANALYSIS BY MD The main problem that arises from the data presented earlier is related to the difference between surface and BPs detected by comparison of electrokinetic and IFC measurements with PL of different structures. As we may conclude, this difference is caused by PL effect on the lipid physical state, especially in the region of lipid polar heads responsible for any changes in dipole potential. The principal role in these effects belongs to lysine contacts with polar groups of lipids and water molecules associated with them. According to the electrokinetic data presented in Fig. 6.8, the surface charge of liposomes becames more positive in wide concentration range where the BP, measured by IFC method with planar BLM, does not change. The latter method “sensed” the lysine adsorption at the membrane surface at about two orders later than zeta-potential approaches to asymptote. It seems that the changes of surface potential are compensated by the opposite variations of dipole component of BP. To support this assumption and to examine the nature of this unexpected effect, computer simulation of the system was done by the methods of MD because they are well suited to test our hypothesis. Bilayer membranes were studied with MD by many authors in different aspects including the electrostatic phenomena at the membrane surface (see review [68] and references therein). According to our experience on bilayers composed of neutral and charged lipids (DPPC and mixtures DPPC/DPPS) in the presence of inorganic cations, the correct comparison between MD and experimental data is achieved when the “electrical” membrane interface is placed at a distance of about 1 nm from the Ca carbon of glycerin bone of the lipid molecules [41]. Lysine effects on the membrane electrostatics and hydration of lipids were studied by MD on systems constructed from 180 molecules of DOPS, about 17,000 molecules of water, and a varied ratio of potassium and lysine cations. The number of cations was about 200 (for sum of potassium and lysine) and anions (Cl) were chosen according to the condition of electroneutrality. Therefore, the amount of free cations in the system does not exceed 20. If this number is assumed to be smaller, the equilibration in the double layer requires too much time. Of note, the ionic strength in the system determined far from the membrane surface appeared

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to be around 150 mM. It is too high in comparison to real conditions in the experiment. No numerical agreement between “digital” and real experiments is expected in this case, but the qualitative conclusions remain important for understanding the nature of the studied phenomena. Boundary and surface potential in systems with different amounts of adsorbed lysine calculated from MD simulation are presented in Fig. 6.9. The electric potential was computed by double integration of charge A BP

Potential increment (mV)

50

SP

40 30 20 10 0 −10

Contribution of water H-bonds to BP (V)

B 0

−0.2

−0.4 H-bonds to C=O PO4

−0.6 −4

−3

−2

−1

Lg [C] (M)

Figure 6.9 Lysine adsorption at the surface of DOPS analyzed by molecular dynamics. (A) Surface potential (SP, opened points) determined as a potential of mean force from Kþ cation distribution in the adjacent water layers. Boundary potential (BP, crosses) determined as a difference between electrical potentials in the center of bilayer and in the bulk of water. (B) Contribution to boundary potential of water H-bonds found and averaged separately for carbonyl groups (black points) and phosphate groups (opened points).

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density, and the difference of the potential found in the center of the bilayer and in the bulk gives total BP value. These calculations reveal sharp changes in the potential exactly in the area of “electrical” interface but the absolute value of potential drop is overestimated. To get a correct value of surface potential, it is necessary to take into account nonelectrostatic components of ion–surface interaction [40]. Particularly, surface potential may be calculated as a potential of mean force from Kþ cation distribution in the adjacent water layers. MD allows us to evaluate the principal tendency in changes of surface potential or total BP at the interface (Fig. 6.9A) and to compare the contribution of any component of the system separately (Fig. 6.9A). In accordance with the experimental data (Fig. 6.8), the increment of surface potential with lysine concentration is larger than that of total BP. The difference between surface and BPs is attributed to dipole potential which shows negative changes in the presence of lysine at the membrane surface. So, special attention has to be paid to the number and orientation of water molecules responsible for the dipole component of BP in the polar region. Indeed, the most expressive changes in profile of total electric potential are observed at 7–10 A˚ from Ca atom of glycerin bone where crucial changes in profiles of diffusion and orientation of water molecules were observed (data not shown). The most probable reason of dipole potential changes in a negative direction is water H-bonds to lipid head groups. We compared two sites of these groups commonly discussed in literature—carbonyl and phosphate. Most authors attribute the dipole effect to carbonyl oxygen. In the case of PL at the membrane surface, the same conclusion was made in Ref. [24], supported by a shift in the vibration band of the C]O group that correlates with the strength of the H-bond to carbonyl oxygen [69]. These results do not exclude the idea that a phosphate group may participate in the dipole effect discussed above, such as was found in Ref. [56]. Moreover, our MD simulations show that a lysine molecule may also act as the H-bond donor. Contact of lysine with lipids is accompanied by a formation of 2.5 H-bonds with head groups including 0.8 H-bonds to phosphate group per each lysine molecule. A different behavior of water around carbonyl and phosphate groups was found in MD simulation and shown in Fig. 6.9B. Both groups are responsible for BP decreasing with lysine at the surface. But the number of H-bonds to PO4 and its contribution to BP became constant at the intermediate lysine concentration but that is not for C]O groups. That is why we prefer to think that the changes in the number of these H-bonds are responsible for the compensation effect observed at lysine adsorption. Surely, this consideration has to be listed in the

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text in more detail and not presented as a solid conclusion because MD techniques cannot supply sufficient proof. We have to note that MD simulation ˚2 detected the average area per molecule of lipid changed from 60 to 58 A with lysine binding to the surface. It may lead to increased rigidity of lipid monolayers similar to that shown in Fig. 6.5. We compared this system to the case of multivalent (Be2þ) and monovalent (Kþ) cations adsorbed at the surface of DPPC/DPPS bilayers. Multivalent cations, but not potassium, decrease the number of water and H-bonds to phosphate group of lipids and change the dipole potential in the experiments [21] and MD simulations [41]. These considerations confirm that the variation of water H-bonds to phosphate group is critical for the changes in dipole potential detected in the experiment.

10. SPECULATIONS ON PL INTERACTION WITH LIPID BILAYERS The studies of different lipid systems in their interaction with PLs are stimulated by related biological phenomena in the cell membranes due to adsorption of charged macromolecules developed for various biomedical applications. A lot of data presented in the literature concern polymer effects on the integrity of biomembranes significant for cell vitality. Another aspect of the problem is a modification of outer cell surface by polymers important for lateral interactions between membrane proteins mediated by lipid matrix. The latter is known as the active participant in enzyme driving processes, and mechanosensitive channels sensitive to the presence of multivalent cations in water media is the best example of nonelectrostatic phenomena correlated with ion adsorption at the membranes. This aspect focused our attention on the polypeptide effect of the electric field distribution at the membrane boundaries and related phenomena that may have a definite role in cell physiology. PL adsorption at the lipid membrane surface induces a number of electrostatic and thermodynamic effects corresponding to changes in the bilayer structure. The second, negative, phases in the BP kinetics during PL adsorption correlate with the compensation of surface potential by opposite changes of dipole potential. The dipole component of BP decreased by PL of various molecule lengths to the same extent because it may be related to caused by direct lysine bases contact with lipid head groups. In contrast, multivalent cations increase the dipole potential. According to MD analysis of the plane of inorganic cation, adsorption is placed at some depth in the

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area of lipid polar heads. Lysine molecules (and PLs) remain at the outer membrane surface and the nature of their dipole effect is restricted by water reorganization only. The rigidity of lipid monolayers increases in the presence of lysine and PL, but polymers of high-molecular weight demonstrate slow kinetics probably due to domain formation. These domains at the surface of lipid bilayer with a high percentage of negatively charged lipid are covered by polymer as the continuous polymer layer with a surface charge density irrelatively on the PL molecular weight in contrast to polymer cluster thickness. As a basis of these phenomena, we assume a specific influence of lysine molecules on the hydration state of lipid polar heads. Special interest is focused on the water orientation and H-bonds that have broken with the phosphate group and communicated with the adsorbed molecules of (poly)-lysine. Unfortunately, we have no theory to describe them in a quantitative manner but only to speculate about some correlations and to make general conclusions. The intensive experiments by the methods presented here may prove or disprove them in the near future.

ACKNOWLEDGMENTS The authors thank Prof. S. Sukharev and Dr. K. Kamaraju, Department of Biology, University of Maryland, College Park, Maryland, for help in the experiments with ITC and Langmuir monolayers. The reported study was supported by RFBR, research project no. 11-0301109a and in part by Program of Presidium of RAS “Molecular and Cell Biology,” and Federal Task Program “Scientific and Scientific-Pedagogic Personnel of Innovative Russia” for 2009–2013 (state contract #8166). MD simulations were performed using Moscow State University supercomputer complex on SKIF “Chebyshev” and “Lomonosov.”

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