Langmuir 1989,5, 390-394
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Fluorescence and Electron Microscopic Study of Lectin-Polysaccharide and Immunochemical Aggregation at Phospholipid Langmuir-Blodgett Monolayers Wolfgang M. Heckl Department of Physics, Technical Uniuersity of Munich, 0-8046 Garching, Federal Republic of Germany
Michael Thompson* Department of Chemistry, University of Toronto, 80 S. George Street, Toronto, Ontario M5S l A 1 , Canada
Helmuth Mohwald Department of Physical Chemistry, University of Mainz, 0-6500 Mainz, Federal Republic of Germany Receiued August 8, 1988. I n Final Form: October 26, 1988 The interaction of phospholipid monolayers with the proteins concanavalin A and IgG antibody was examined by film balance measurements and by fluorescence and electron microscopies. Concanavalin A affects the size of lipid domains through preferential arrangement at the boundary between ordered domains and fluid environment. The aggregated protein present in structures formed by bonding with dextran both alters the shapes of lipid domains and increases monolayer viscosity by participation in a network that interconnectsdomains. The uniform shape of the aggregates (about 40 nm in size) and regularity of interaggregate distances can be attributed to the effect of long-range electrostatic forces. Although less pronounced, similar results to the above were observed for antibody-antigen interactions.
Introduction Protein aggregation, as observed in bilayer lipid membranes (BLM), plays an essential role in biological cellular activity. Such a process has been invoked to explain transient electrochemical behavior observed in experimental BLM systems.ll2 In that work, sudden, but reversible, changes in membrane ion permeability caused by interaction with lectin-glycogen and antibody/antigen pairs were speculatively ascribed to perturbation of the membrane surface dipole potential by the formation of protein-based aggregates a t the membrane surface. Analogous spontaneous pulsing of electrical potential has been observed for measurements made across LangmuirBlodgett films.3 Furthermore, the surface potential for phosphatidylcholine/cholesterolmonolayers was measured by a nonconducting electrostatic probe for dynamic subphase infusion of lectin-polysaccharide and immunochemical c o m p l e x e ~ . ~This system produced transients in dipole potential that were attributed to local alteration of lipid dipole orientation caused by the formation of aggregates a t the monolayer/water interface. The conclusions derived from the above experiments are based on the assumption that protein is bound a t the bilayer or monolayer "surface". A direct technique for the study of such a process is Langmuir-Blodgett fluorescence micro~copy.~ A typical application is the study of the quantitative reconstitution of proteins associated with photosynthetic processes in a monolayer membrane.6 In the present paper, we examine the interaction of concanavalin A (con A) and the protein-dextran combination with monolayers formed from dipalmitoylphosphatidylcholine (DPPC) by both electron and fluorescence microscopy. A small number of similar experiments were also conducted with a typical antibodylantigen pair. Experimental Section Reagents. The phospholipid L-cu-dipalmitoylphosphatidylcholine (DPPC), concanavalin A (con A, purity ==99%, MW i=
*Author t o whom correspondence should be addressed. 0743-7463/89/2405-0390$01.50/0
105 000)and fluoresceinyl-labeled con A (containing 4-8 mol of FITC per mol of protein), dextran (MW = lOOOO), and goat anti-rabbit IgG and rabbit anti-goat IgG were obtained from Sigma Co., Taufkirchen, FRG, and used without further purification. The antigen-specificantibody, used in this work, was isolated from goat anti-rabbitIgG antisera by immunospecific purification and was determined to be immunospecific for rabbit antigoat IgG (Sigma product information). The dye, dipalmitoyl(nitr0benzooxadiazolphosphatidy1)ethanolamine(DP-NBD-PE),was obtained from Avanti Polar Lipids, Birmingham, AL. The water used was distilled,deionized and filtered with a Milli-Q system. Apparatus. The fluorescence microscope and film balance have been described previ~usly.~ Excitation of fluorescence is achieved via a water immersion objective lens placed in the bottom of the Langmuir-Blodgett trough. Emitted light is imaged on a proximity focus image intensifier television camera with textures being photographed directly from the screen. The surface potential of the monolayers transferred to a solid substrate was measured with a recently described setup' by using a vibrating plate capacitance method. Electron microscopy was carried out on a Phillips Model EM 400T microscope. Platinum shadowing was effected by using a Balzers BAFD 400 evaporation chamber. Procedures. Mixtures of DPPC and dye containing 1mol % of the latter in 3:l v/v chloroform/methanol mixed solvent were spread on the surface of the water containing lo-* M Mn2+and Ca2+. After the monolayer was compressed to a pressure of approximately 5 "em-', the protein (50 WLof an aqueous solution at a concentration of IO4 M) and, if desired for aggregation studies, the dextran solution M) were spread on the fluid monolayer surface by using a syringe as described previously.8 Similar (1) Thompson, M.; Krull, U. J.; Bendell-Young, L. I. Bioelectrochem. Bioenerg. 1984, 13, 255. (2) Krull, U. J.; Thompson, M. Biochem. Biophys. Res. Commun. 1986,141, 912. (3) Ishii, T.;Kuroda, Y.; Omochi, T.; Yoshikawa, K. Langmuir 1986, 2, 319. (4) Thompson, M.; Wong, H. E.; Dorn, W. H. Anal. Chim. Acta 1987, 200, 31. (5) Losche, M.; Mohwald, H. Reu. Sci. Instrum. 1984, 55, 1968. (6) Heckl, W. M.; Losche, M.; Mohwald, H. Thin Solid Films 1985, 133, 73. (7) Heckl, W. M.; Baumgartner, H.; Mohwald, H. Thin Solid Films, in press. ( 8 ) Heckl, W. M.; Losche, M.; Scheer, H.; Mohwald, H. Biochim. Biophys. Acta 1985, 810, 73.
0 1989 American Chemical Society
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Phospholipid Langmuir-Blodgett Monolayers 50
261
I
I
I
Y
0
0.4
0.6
0.8
0
1.0
m o l e c u l a r a r e a A [nm2/molecule DPPCl
Figure 1. Surface pressure as a function of molecular area per lipid for DPPC monolayer before (a) and after spreading of an aqueous protein solution (containing a small amount of detergent solution remaining after dialysis) leading to a layer containing con A in a protein/lipid ratio of 1/100 (b),con A in a protein/lipid ratio of 1/100 and additionally lod M dextran (c), and anti-rabbit IgG in an antibody/lipid ratio of 1/100 (d). T = 25 "C, pH 6. experiments were conducted with IgG species (0.025 mg in 100 p L of water). For study by electron microscopy, a monolayer containing lipid and con A-dextran complex was transferred onto a glass slide by the typical Langmuir-Blodgett dipping technique. The slide was removed from the subphase at a speed of 0.3 cmamin-' while the lateral film pressure was held constant at 15 "am-'. Low-angle (15") platinum shadowing was performed in an evaporation chamber to deposit Pt clusters of about 1-nm diameter. The platinum layer thickness is about 0.5 nm. In order to further stabilize the film, 100-8, carbon was vacuum-deposited perpendicularly from the top. In order to separate the film from the glass slide, the sample was immersed into hydrofluoric acid ( 5 vol %), where the glass slide was dissolved and the replica, which floats to the surface, could be picked up with an electron microscopic grid. Subsequent to deposition onto electron microscopic grids, the replica was examined by transmission electron microscopy at magnifications between 400X and 200 OOOX. For the study of surface potential, the lipid monolayer containing protein-con A complexes was transferred onto HF plasma cleaned silicon wafers.
Results and Discussion Protein-Containing Monolayers at the Air/ Water Interface. Phase Diagrams and Fluorescence Microscopy. The phase diagram of a pure DPPC monolayer exhibits a pressure-area isotherm (a-A curve) with a two-phase coexistence region a t pressures over a , . This region corresponds to the pronounced main phase transition from a two-dimensional liquid to a liquid crystalline phase state, where a gel phase (solid domains) is in coexistence with a continuous liquid phase (Figure 1, curve a). After interaction with con A or antibody, an expansion of the isotherms is observed, as shown in curves b and d of Figure 1, respectively. The molecular area increase caused by the incorporation of these species can be used to estimate the amount of protein present within the monolayer. For example, a typical increase of 0.1 nm2 in the molecular area (measured a t the main phase transition a t a, in Figure 1 curves (a and c) corresponds to a protein/lipid ratio of 0.1 nm2/12.5 nm2 = 0.008, assuming a con A monomer cross section of about 12.5 nm2 (according to crystallographic ~ t u d i e s . ~ In ) comparison to the initial amount of spread protein (1mol %), this leads to a typical incorporation ratio up to about 80%. This result, which indicates penetration of protein into the lipid layer rather than just adsorption from the subphase, can be explained in view of the fact that the protein possesses a hydrophobic pocket, which allows its interaction with hydrophobic moieties in monolayersg and membranes.1° Addition of (9) Edelman, G. M.; Wang, J. L. J . Biol. Chem. 1978, 253, 3016.
0.5
1
time t lhl
Figure 2. Film pressure as a function of time following a pressure decrease after expansion of a DPPC monolayer containing con A and dextran as in Figure IC. dextran, which causes the formation of cross-linked aggregates with con A in the presence of Mn2+and Ca2+," produces a a-A curve slightly shifted to higher pressures (Figure 1, curve c), whereas the addition of the polysaccharide alone to the monolayer does not significantly alter the isotherm. Further compression of a monolayer containing con A or con A-dextran complexes to about 40 "em-' results in an isotherm typical of pure DPPC with the area occupied by a lipid molecule being about 0.45 nm2. This loss of monolayer-embedded protein a t higher film pressures has been observed previously12-16 and indicates that incorporation of protein requires preparation of these samples under sufficiently low pressure. Penetration was hindered when the film was tightly packed, as compared to a looser film, and only adsorption was possible. On expansion of the film, the reverse process, i.e., the penetration of the proteins into the film, could be studied. Figure 2 shows a typical penetration isotherm, where the new equilibrium pressure a ( t = a) is reached in an exponential pressure-time dependence. The time constant of this process is in the order of 1h. Assuming a reasonable diffusion constant of about lo4 c m 2 d , the path length of a three-dimensional diffusion of a protein in the bulk is in the order of 1 mm. This could be an indication that the protein slowly diffuses from a bulk zone very close to the monolayer surface. An alternative explanation for this slow pressure increase could be a readsorption of the protein, which may only reflect a time-dependent structural change. Such binding to the lipid head groups could also cause rearrangement of the DPPC dipoles. However it is reported (cf. ref 14) that the perturbation induced in the lipid matrix by binding of a protein is very small and localized. Comparing these two possible mechanisms, we believe that the fact that the pressure change occurs on a relatively long time scale favors an explanation by a diffusion-limited penetration process rather than a process limited by a structural change of the protein. The reference experiment, Le., the expansion of the film without protein, shows no slow pressure increase with time but a reversible monolayer phase behavior along the isotherm. Additional information on the nature of the lipid-protein interaction can be obtained from a comparison of fluorescence micrographs. Figure 3a depicts the rela(10)Goldstein, I. J.; Reichert, C. M.; Misaki, A. Ann. N. Y. Acad. Sci. 1974, 234, 283.
(11) Harrington, P.C.;Wilkins, R. G. Biochemistry 1978, 17, 4245. (12) Heckl, W. M.; Zaba, B. N.; Mohwald, H. Biochim. Biophys. Acta 1987, 903, 166. (13) Heckl, W. M.; Mohwald, H. J. Mol. Electron. 1987, 3, 67. (14) Wilkinson. M. C.:Zaba. B. N.: Tavlor. D. M.: Laidman, D. L.: Lewis; T. J. B i o c h . Bcophys.'Acta 1986,- 857, 189. (15) Teissie, J. Biochemistry 1981, 20, 1554. (16) Quinn, P.J.; Dawson, R. M. C. Biochem. J . 1970, 116, 671.
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.Figure 4. Fluorescence micrographs OS a DPPC monolayer containing 1%DP-NBD-PE and additionally 1 mol % goat anti-rahhit I& antibody and 1 mol % rabbit IgG antigen. Again cross-linking is evident on the water surface (a) as well as in the layer transferred t o a SiO, substrate (b).
Figure 3. Fluorescence micrographs: (a) DPPC monolayer containing 1%DP-NBD-PE in the coexistence region (T = 12 mN.m-', T = 25 "C). (b) Same film as in part a with additional con A in a proteinjlipid ratio of 1/100 shortly after incorporation. (c) DPPC monolayer containing 1 mol % fluorescence-labeled protein con A. (d) Same film as in part b with additional M dextran after a short time. (e) Same film as in part d after 1h. Cross-linking 'ropes" between DPPC domains can be seen in gray connecting the dark lipid domains. tively regular shape and size of gel-phase domains of a DPPC monolayer (dark) among a bright fluid lipid phase containing DP-NBD-PE. The addition of con A or antibody results in an inhibition of crystalline domain growth (Figure 3b). This effect can be attributed to a reduction of edge free energy on accumulation of protein at the interface between liquid crystalline domains and fluid areas. Such a process will favor the production of a large number of smaller domains at the expense of a small number of big domains. Support for this argument can be derived from Figure 3c, which shows a fluorescently labeled con A protein causing the appearance of halos at the domain boundaries. The influence of lectin and polysaccharide was first examined in an experiment where the film was compressed shortly after the addition of the reagents (Figure 3d). As can be seen by comparison with Figure 3a and 3b, the shape of the domains is grossly changed. Since this effect is not caused by the singular addition of each reagent, we conclude that it must be associated with the early stages of complex aggregate formation. Compression of the film will result in an increased protein concentration in the zone immediately surrounding the domains. Complexation with dextran allows the bridging of domains that appear as a growth of domain dendrites. Moreover, the greater number of edge tension-reducing proteins at the liquid crys-
tal-fluid phase boundary could result in the formation of dendritic domains. In a second experiment, where a significant time was allowed to elapse (about 1 h) before compression (Figure 3e), cross-linked con A-dextran "ropes" connecting solid lipid domains are clearly evident. The lectin has been employed in these experiments as a model for immunochemical reactions. Experiments conducted with antibody-antigen pairs gave similar results. For example, Figure 4a shows an analogous cross-linking effect when antigen is added to a monolayer to which antibody is incorporated. Protein-Containing Monolayers on a Solid Support. Fluorescence Microscopy a n d Surface Potential. The cross-linked structures were successfully transferred to a solid substrate for examination by fluorescence microscopy and by surface potential measurements. Figure 4b shows a fluorescence micrograph of a DPPC monolayer containing DP-NBD-PE and the cross-linked con A-dextran complex transferred onto the silicon wafer a t 15 mN.m-' film pressure, where the monolayer is in the phase coexistence region. The domain stricture is not grossly altered, so cross-linking can be regarded as a stabilizing effect for a film transfer (like polymerization). A comparison between the lipid-protein layer and the pure monolayer shows that the surface potential of the monolayer-containing protein is reduced from about +400 mV (Vfiptd)to +200 mV, indicating an overall excess dipole moment of the protein of opposite direction to that of the lipid. For a rough estimate we consider the following equation: Viipid-protein vlipido.9 + vprot%in0.1 VIipidTmtein is the surface potential of the monolayer-containing proteins, and !IEpid and Vpr,, are that of the lipid film and the proteins, respectively. The estimated area fraction of the lipid is 0.9, and 0.1 is that of proteins according to the area increase of roughly 10% after protein
Langmuir, Vol. 5, No. 2, 1989 393
Phospholipid Langmuir-Blodgett Monolayers indentation
protein
distance iiim)
a Figure 5. (a) Electron micrograph of a con A-dextran complex containing DPPC monolayer (magnification = 10000). Transfer from the phase coexistence with a protein/lipid ratio of l/lOO hy weight. (b) Correspondingpair correlation function and optical diffraction pattern. incorporation (cf. Figure 1). This leads to a protein contribution to the surface potential of about -1600 mV. With the Helmholtz equation V = rc/(e,eA
(e,
C.V.m-' and e, = relative dielectric constant), which describes the surface potential of a monolayer depending on mean molecular area A (about 50 nmz/con A tetramer), one derives a protein dipole moment perpendicular to the surface of p/er = 7.65 X ICm C m = 200 D. Although this is only a very rough estimate due to the uncertainty about the actual protein fraction in the surface, this value is not unrealistic. It corresponds to the displacement of an elementary charge over 5 nm. Similar values are reported for a number of other protein~.'~.'~ Electron Microscopy. Additional and more detailed information on the cross-linked con A-polysaccharide complex distribution is obtained from electron micrographs. Figure 5a shows an overview of the lateral arrangement of a protein-dextran complexed area. One interesting feature is the occurrence of a homogeneous array of protein aggregates (number about 10l0/cm2,size about 40 nm, and height about 3 nm) with rather equally spaced nearest-neighbor distances. As a quantification of the spatial distribution, Figure 5b gives an analysis of the pair correlation as well as an inflection pattern of Figure 5a, taken with an optical diffractometer. Only distances larger than about 200 nm are evident, and a very regular size of protein aggregates is derived from the diffraction image corresponding to the first range of destructive interference. In principle, the pair correlation function gz(r) of Figure 5 can be converted into intermolecular potential according to g2(r) = e-W(r)/kT = 8.85 X
(11) MeClellan, A. L. In Tables Of Experimental Dipole Moments; Freeman and Company: San Francisco, 1963. (18) Ondey, J. L. In Plateins,Amino Acids and Peptides (IS Ions ond Dipolar Ions; Cohn, E. J. Edsall, J. T., Eds.; Litton Educational Publishing, 1971.
Figure 6. (a) Electron micrograph of a con A-dextran complex (magnification = 45000) containing DPPC monolayer. (h) Schematic view of Dossihle structure of con A proteins adsorhed to the lipid layer and cross-linked hy dextran. W is the mean intermolecular potential for an ensemble of N particles, where one averages over the influence of the remaining N - 2 particle^,'^ and k is the Boltzman constant. In statistical theories of liquids, one often uses an approximation of a 6-12 potential (Lennard-Jones potential): w(r) = const(ro/r)'z - (ro/d6 The first term corresponds to a hard-core repulsion and is in our case not due to volume exclusion, because ro = 200 nm (cf. Figure 5b) is much larger than the size of a protein aggregate (= 40 nm). Therefore we assume a long-range electrostatic dipolar interaction between the protein clusters. This is reasonable in view of the large dipole moment attributed to the protein. Because g2(r) is not determined precisely enough, we cannot experimentally distinguish between a repulsive or an attractive potential leading both to qualitatively the same g2(r)curve (see ref 20). One reason for this is that a detailed analysis requires the investigation of a few thousand points whereas here only about 500 are available. Also, the exponents of W(r) are not necessarily 6 and 12; e.g., Braun et al.I5 simulated membrane protein interactions using a 6-4 pair potential. Another explanation for the observed distribution could be (1) a lipid-mediated protein-protein interaction resulting from distortion of the lipid structure near the protein. This effect could he characterized by a fluctuation of the degree of lipid chain orderingz' leading to an exponential distance dependence of the interaction energy for large protein distances and an attractive force between two proteins. (2) Whereas the former mechanism is more (19) de Boer, J. In Reports on PIogress in Physics; The Physical Society: London 1949; p 12. (20) Braun, J.; Abney, J. R.;Owieki, J. C. Biophys. J. 1987,52, 421. (21) Sackmann, E. InBiaphysik: Hoppe. W., Lahmann, W., Markl, H., Ziegler, H., E&.; SpringerVerlag: WeSt Berlin, 1982.
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valid for short-range forces, a long-range interaction could also be caused by an elastic deformation of the lipid layer due to protein i n c ~ r p o r a t i o n . ~ This ~ ~ ~protein-induced ~ deformation field could lead to interaction energies in the order of kT for a distance of about 100 nm whereby the intermolecular interactions are long range and proportional to l / r so that no exponential distance dependence is expe~ted.*~ Another interesting aspect of Figure 6a is the existence of small indentations in the replica, which can be distinguished from protruding proteins by the opposite direction to which their platinum shadow points in comparison to that of the protein. The number of these randomly distributed indentations is about 101°/cm2with a diameter of about 5-10 nm, and the occupied area is about 1% of the total monolayer area. We believe that these small indentations point to a con A-dextran network. The dextran serves as an anchor and thus disturbs the order (e.g., tilt of molecules) in the lipid layer, resulting in these indentations (cf. schematic, Figure 6b). For this reason a netlike protein aggregate structure with distinct minimum distances is possible. A similar case is discussed in ref 25 where the binding of spectrin filaments to an erythrocyte is explained by coupling to integral membrane proteins and adsorption to the lipid by electrostatic binding. An alternative explanation involving a dynamic process in building and destroying the aggregates in the light of their natural protein structure and function cannot be e x ~ l u d e d .Support ~ for this argument is evident in the appearance of smaller and nonaggregated protein complexes in the left part of Figure 6a. Since these micrographs show a “frozen” state of the monolayer, this kind of dynamics cannot, of course, be observed directly. The second surprising fact is the regular ringlike cylindrical aggregate shape of these protein complexes covering an area corresponding to about 30 proteins (cf. Figure 6a). Again, this could be explained by the role of electrostatic dipolar forces leading to an alignment of proteins as a linear array. The existence of in-plane dipole moments (22) Mouritse; 0. G.; Bloom, M. Biophys. J. 1984, 46, 141. (23) Riegler, J.; Mohwald, H. Biophys. J . 1986, 49, 1111. (24) Sackmann., E.:, Kotulla., R.:, Heiszler. F. J. Can. J . Biochem. Cell B i d . 1986, 62, 778.
(25) Sackmann, E.; Sui, Sen-fang; Wirthensohn, K.; Krumow, T. Biomembrane and Receptor Mechanism; 1987, 7, 97.
could then even cause an antiparallel arrangement of two such arrays, forming two parallel half-sides of the elongated ring.
Conclusions In the present work, we have demonstrated that crosslinking phenomena involving lectin-polysaccharide and immunochemical systems can be detected in a phospholipid monolayer at a microscopic level. By using deposition onto a surface for study by electron microscopy, we found a surprisingly regular macroscopic protein superstructure in addition to very regularly shaped aggregates. Although the distributiork correlation function is at hand, we are so far not able to distinguish between the different possible mechanisms responsible for the structural arrangement and distribution of the proteins, which must reflect the forces acting within the membrane. Because we observe a large excess dipole moment for the protein con A, we believe that electrostatic interactions play an essential role besides the elastic ones. Further experiments with higher protein concentration should shed more light on this problem. In addition to the above, we have successfully transferred, in an intact fashion, monolayer films to which cross-linked proteins are attached by relatively weak forces. Such films are becoming increasingly employed in the development of microelectronic structures26and biosens o r ~ . Moreover, ~~ further study using the techniques described here should enable an explanation of dynamic processes such as functional aggregation of macromolecules. Acknowledgment. We appreciate helpful discussions with M. Egger and P. Karg (Technical University Munchen) and the cooperation of H. Baumgartner (Universitat der Bundeswehr Munchen) in performing the surface potential measurements. The work was supported by the Deutsche Forschungsgemeinschaft through SFB 143. Also, M.T. is very appreciative for the opportunity and funding from the Universitat der Bundeswehr to be able to contribute to this work. Registry No. DPPC, 63-89-8; dextran, 9004-54-0. (26) Roberts, G. G. Contemp. Phys. 1984, 25, 109. (27) Thompson, M.; Krull, U. J. TrAC, Trends Anal. Chem. 1984,3,
173.
