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Ordered Protein Arrays as Mesophases H. Haas and H. Mohwald’ Institute of Physical Chemistry, University of Mainz, D 55099 Mainz, FRG, and Max Planck Institute for Colloids and Interfaces, D 12489 Berlin, FRG Received September 10,1993. In Final Form: November 29,199P Domains of orientationally ordered streptavidin molecules bound to biotinylated amphiphile monolayers at the airlwater interface can be compressed reversibly by a factor of 2 without destroying the order. By X-ray reflectivity it is shown that the water content of the protein domainsat low lateral pressure corresponds to that of 2D crystalsstudied previously after transfer on solid support. The domains are highly compressible since 50 vol % of water can be removed upon compression. The results support the view that the protein forms a mesophase and not a crystallinephase. Studies of the protein crystals have yielded much insight in understanding biological problems, but the availability and resolution are limited as the majority of the systems of interest does not crystallize readily. One new possibility is the growth of 2D crystals at surfaces. Crystallization of proteins at interfaces is an interesting problem with perspectives in biophysics, biology, and general physics: Binding to an interface may preorient a protein and therefore enable structural studies with two-dimensional protein crystals. On the other hand these are lowdimensional structures and the question arises if long range order is formed at alL1 Streptavidin (SA), a tetrameric protein crystallizing with C222 symmetry6 strongly binds to various monolayers of biotinylated lipid.3~~ By fluorescence microscopy with fluorescently labeled protein, one can observe domains of uniform polarization, proving that dye probes attached to the protein are uniformly These domains could be transferred on electron microscope grids and after staining they yielded many diffraction spots to enable high resolution structural analysis.2 Although the proteins in these aggregates exhibit high positional order, they have only low packing density. In this report we show that such aggregates can be compressed and expanded by about a factor of 2 maintaining the orientational order detected by fluorescence microscopy. We assume water being squeezed out of the protein domains by decrease of the protein spacings. This would support a liquid-crystalline-like structure of the proteins instead of a crystalline one. In the experiment presented here, SA was bound to monolayers of binary mixtures of an amphiphilic polyacrylate (Figure 4c) with betain-caproyl-DPPE (B-CapDPPE, Figure 4c). By the use of binary mixtures of biotinylated lipid with an inert matrix lipid, the molecular area per biotin group can be selected to a high accuracy. The molecular area of SA binding to the biotin can be changed by compression or expansion of the lipid monoAbstractpubliehedin Advance ACSAbstracts, February 1,1994. (1)Halperin, B. J.; Nelson, D. R. Phys. Rev. Lett. 1978,41,8214. (2)Daret,S.A.;Ahlere,M.;Meller,P.H.;Kubalek,E. W.;Blankenburg, R.; Ribi, H. 0.;Ringsdorf, H.; Komberg, R. D. Biophys. J. 1991,59,387. (3)Blankenburg, R.; Meller, P. H.; Ringsdorf, H.; Salesse, C. Biochemistry 1989,28,8214. (4) Ahlers, M.; Blankenburg, R.; Haas, H.; Mbbius, D.; Mbhwald, H.; Milller, W.; Ringsdorf, H.; Siegmund, H.-U. Adu. Mater. 1991,3, 39. (5)Hendrickeon, W.A,; Pihler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.;Phizackerley, R. P. h o c . Natl. Acad. Sci. U.S.A. 1989,86,2190. (6) Als-Nielsen, J. Phys. A 1986,140,376. (7)Ale-Nielsen, J.; Mtjhwald, H. In Handbook of Synchr. Rad. Ebashi, S., Koch, M., Rubenstein, E., Eds.; Elsevier: Amsterdam, 1991;Vol. 4. (8)Baas, H.; Mbhwald, H. Colloids Surf., B 1993,1, 139-148. (9)Vaknin, D.;Als-Nielsen, J.; Piepenstock, M.; Lhche, M. Biophys. J. 1991, 60, 1545. Vaknin, D.;Kjaer, K.; Als-Nielsen, J.; Lbsche, M. Biophys. J . 1991, 59, 1325. Vaknin, D.; Kjaer, K.; Ringsdorf, H.; Blankenburg, R.; Piepenstock, M.; Diederich, A.; Lbche, M. hangmuir 1993,9,1I71-1174. @
layer. Due to its bulky headgroup the polymer displays liquid expanded (LE) phase over a wide ran e of area starting from ca. 70 A2 per alkyl chain to 25 i2/chain. The amphiphilic polyacrylate containing 7% ’ of B-CapDPPE was spread in a Langmuir trough on the surface of Millipore filtered water containing 0.5 M NaC1. The protein was injected into the subphase at a surface pressure of 20 mN/m to reduce unspecific insertion of protein into the monolayer. Then the monolayer was expanded to ca. 5 mN/m for adsorption. After waiting for at least 3 h, we performed X-ray reflectivity measurementsof that system as a function of molecular area of the lipid. The reflectivity is measured as a function of incidence angle a with respect to the surface where the reflectivity is given by the master
R is the reflectivity and RF the Fresnelreflectivity expected for a sharp interface. pw is the electron density of the subphase, p’ = dpldz is the electron density gradient normal to the surface, z is the coordinate along the normal, Qz = (4a/h) sin(a) is the normal wave vector transfer for, wavelength X and incidence angle a. Figure 1 shows the results of X-ray reflectivity measurements of the protein bound to the polymedbiotin monolayer at low surface pressure (a) and after compression and expansion of that monolayer to different molecular areas of the lipid (b-e). One realizes a shift of the extrema to lower Q on compression and an increase in the amplitudes of the oscillations. Also the number of the discernable minima and maxima increases. Expansion and, several hours later, recompression yields basically the same curves as before at these positions. We analyzed the data using a model of three stratified laterally uniform layers: one layer for the hydrocarbon chains of both lipids,one for their electron-rich headgroups, and one for the protein plus water. Keeping the intensity constant, three lengths and electron densities and two roughness parameters (taking the protein roughness different from the lipid roughness) were fitted. The numbers we got for thickness and electron density for the two lipid slabs were consistent with data we found for pure lipid monolayers in previous experiments. Figure 2a shows the fit parameters of length and electron density for the protein slab. Both, electron density and length increase slightly but by far not enough for the conservation of substance in the protein layer according to the compression ratio. Figure 2b displays the number of electrons for the lipid (head + tail) and for the protein under the respective molecular area of a lipid chain. For the lipid,
0743-7463/94/2410-0363$04.50/00 1994 American Chemical Society
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364 Langmuir, Vol. 10, No. 2, 1994 50
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Figure 1. X-ray reflectivity and fits normalized to the Fresnel reflectivity (R/RF)as a function of normal wave vector transfer Q1for SA bound to a polymer monolayer at the aidwater interface at different areas of the hydrocarbon tails. The ordinate is exact for the lower curve and subsequently shifted by one for the respective upper curves. Curve a was measured more than 3 h after protein injection. The film was then compressed to lower molecular areas and reflectivity measurements were performed (b,c). After expansion to the area of c and waiting for 50 h, curve e was measured. For that experiment the subphase contained 0.5 N NaCl, the SA was injected to give a concentration of 5 X 10-8 M. the electron number matches the number calculated from the amount of spread solution and is roughly constant, whereas in the protein layer the number of electrons is decreasing continuously on compression. This means there must be some loss of material into the subphase. Reflectivity curves taken again at high molecular area after maximal compression yielded the same results as during the compression run, particularly there was no change in thickness and electron density of the protein layer. We deduced that water must be squeezed out of the protein slab, a desorption of protein from the surface would very unlikely be reversible. Figure 2c shows the number of water molecules per protein for different presumed volumes of the SA as a function of the electron density. Taking Vprot= 65 000 A3 as an example, the protein slab contains in the equilibrium state 3000 water molecules per protein and on compression 1700 water molecules are squeezed out. To get further information on that phenomenon we performed analogousfluorescence microscopy experiments using fluorescently labeled protein. In order to keep track Df a defined area of the monolayer, a circular spot of the protein aggregates was bleached. Figure 3 shows the proteidlipid monolayer before and after bleaching and at serveral stages of compression and expansion for two polarizer positions and without polarization. Obviously the bleached spot and also the polarized domains (see nrrow) of the protein are shrinking on compression. The gpot is deformed in the direction of compression. Polarization and shape of the protein domains are maintained. 3n expansion the domains regain almost their original size.
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Figure 2. Model parameters obtained from fitting a three slab model to the reflectivitydata: (a,top) length and electrondensity of the protein slab vs area of the hydrocarbon chains; (b,middle) number of electrons Ni in the slabs normalized to the chain area A, as derived from Ni = p&iA; (c, bottom) number of water molecules n per protein v8 electron density Ph in the protein slab. n is calculatedpresuming differentdry volumina V, of the protein fromtherelationp = (Np+nN,)/(V,+ nV,)withp,theabsolute electron density o! the protein slab, Npthe number of electrons per (dry)protein,N , the number of electrons per water molecule, and V, the volume of a water molecule. The bold part of curve c indicates the change on compression as undergone in this experiment. The fluorescence micrographs show, that for all stages of compression and expansion, the protein layer is homogeneous without any separation of the protein dmains. This is in accordance with the reversibility of the X-ray curves on expansion and recompression (Figure lb-d) (a film consisting of an area fraction of lipid bound protein and one of pure lipid should yield a significantly different reflectivity curve to the case of laterally uniform layers as modeled here).8 The numbers in Figure 3 for the compression ratio and the relative area of the bleached spot are in good agreement for all points, only the relatively small area of the spot after expansion corresponds to a shift of the isotherm to smaller areas and may be the result from a minor loss of material. For a molecular understanding of our results Figure 4a shows the packing of the streptavidin derived from electron diffraction2 in side and top view. According to this model there is a very loose packing of the streptavidin with an area per molecule of 3600 A2. Taking the protein volume as before to V, = 65 000 A3,there should be 2800 water molecules per protein. Compression could lead to a packing as sketched in Figure 4b with a smaller unit cell for the protein containing much less water. Maintenance of the global shape and orientation of the protein domains can be supported by the viscosity of the polymer matrix
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1 m05 1.87 Figure 3. Fluorescence micrographs of fluorescently labeled SA bound to the monolayer on compression and expansion: row a, monolayer with unpolarized emission and with emission polarized in two perpendicular orientations; row b, same position of the film after bleaching a circular spot through an aperture by the excitation light of the microscope; (c, d) compression; (e) expansion. Far right: compression ratio of the monolayer compared to the relative area of the bleached spot. The arrow indicates a individual domain which can be tracked along the whole run of compression and expansion. Subphase conditions were the same as for Figure 1.
above and the resulting hindered mobility of the biotin anchors. We should add that the observed variations in protein density are not specificfor the present system. Subsequent X-ray and neutron reflectivity measurements of SA bound to different phospholipid monolayersyielded a dependence of protein packing density on steric and dielectric constraints of the head group^.^ Repulsive interaction of
disordered aliphatic tails may influence the protein aggregation. This means that the lattice found from electron diffraction of transferred (and hence recrystallized) films of sharp edged domains, which are formed if only biotinylated lipid of low density is present at the interface2s4is only one of a wide range of possible structures. Protein domains being compressible like a liquid, maintaining fluorescence polarization (i.e. long range
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366 Langmuir, Vol. 10, No. 2, 1994
x=y=l
b
Figure 4. Side and top view of SA binding to the monolayer: (a) sketches the packing as derived in ref 2; (b) suggesbd from this work. The lipids used are shown in part c.
orientational order) over distances larger than 10 pm are not classical crystals, but rather smectic liquid crystals. The positional ordering could be comparatively low for that case. A proof for that model can be obtained by in situ X-ray diffraction experimenb which hopefully become feasible with protein domains in the near future.
Acknowledgment. We thank U. Siegmund, Bayer AG, for the generous gift of the amphiphilic polymer and C. A. Helm for many stirnulatingand illuminatingdiscussions. The work was supported by the Bundesministerium fiir Forschung und Technologie (BMFT) and by the German Israeli Foundation (GIF).
