Langmuir 1993,9, 1171-1174
1171
X-ray and Neutron Reflectivity Studies of a Protein Monolayer Adsorbed to a Functionalized Aqueous Surface David Vaknin,+ Kristian Kjaer,x Helmut Ringsdorf,s Rainer Blankenburg,$ Michael Piepenstock, 1 Anke Diederich,l and Mathias Liische'J Physics Department, Ames Laboratory, Iowa State University, Ames, Iowa 50011, Physics Department, Rise National Laboratory, DK-4000 Roskilde, Denmark, and Institute of Organic Chemistry and Institute of Physical Chemistry, University of Mainz, 0-6500 Mainz, Germany Received January 5,1993. In Final Form: March 23, 1993
We report the structural organization of a protein, streptavidin, specifically bound at aqueous surfaces to lipid monolayers functionalized by biotinylated head groups. X-ray and neutron reflectivity data with HzO and DzO as subphases are consistent with the formation of homogeneous monomolecular protein layers (thickness d,, = 42 f 2 A) directly underneath the lipid. A new approach of analyzing all four data seta in terms of one general model reveals the dry volume of the protein, ita average lateral density at the interface, and the amount of water interpenetrating the protein film.
Introduction The interactions of proteins with membrane surfaces govern many phenomena critical for the formation and survival of living cells, such as structural reinforcement, signal processing, and self/non-self-discrimination. In materials science, the characterization and the control of the structural organization of proteins at interfaces are of immediate importance for the design of novel devices for analytical or sensoric applications. For the investigation of the underlying self-organization principles, functionalized Langmuir monolayers may be exploited. The structure of these molecular films on aqueous surfaces may be controlled through the manipulation of physical and chemical variables (molecular structure and lateral density of the constituent molecules, temperature, pH and ionic strength of the subphase, etc.). X-ray and neutron reflectometry are well established as powerful surface characterization techniques.lP2 The recent development of reflectometers specifically built to investigate free gasliquid surfaces has provided new possibilities for the characterization of the structure of liquid interfaces on the molecular length scale.3 X-ray and neutron reflectivity methods as well as in-plane diffraction techniques have been widely utilized for structural studies of amphiphilic monolayers on water surfaces and have yielded valuable insight into the properties of these system^.^ We have applied these techniques for the first time to investigate protein recogition processes at functionalized Langmuir monolayers. As a model system for our investigations we used the well-known streptavidin-biotin ~ y s t e m . ~Streptavidin (SA), a tetrameric protein of -55 kD extracted from Streptomyces avidinii, has an exceptionally high affinity to biotin with a binding constant comparable to that of
* To whom correspondence should be addrewed.
+ Iowa State University.
t Ria0 National Laboratory. 0 Institute of Organic Chemistry, University
of Mainz. of Physical Chemistry, University of Mainz. (1) Tidawell, I. M.; Ocko, B. M.; Pershan, P. S.; Wasserman, S. R.; Whitesides, G. M.; Axe, J. D. Phys. Rev. B 1990,41,1111 and references therein. (2) Ruseell,T. P. Mater. Sci. Rep. 1990,5,171 and referencestherein. (3) Jacquemain, D.; Grayer Wolf, S.;Leveiller,F.; Deutach, M.; Kjaer, K.; Als-Nielsen, J.; Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1992,31,130. Als-Nielsen,J.; Kjaer, K. In Phase Transitions in Soft Condensed Matter; Riste, T., Sherrington, D., Eds.; NATO AS1 Series B 211; Plenum Press: New York, 1989; p 113. Als-Nielsen, J.; Mdhwald, H. In Handbook on Synchrotron Radiation; Ebasi, S., Koch, M., Rubenstein, E., Eds.; Elsevier: Amsterdam, 1991; Vol. 4, p 1.
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covalent bondsS4It can bind four ligands, two at each of two opposing faces. Recent fluorescence microscopy studies showed that SA organizes in macroscopic domains underneath aqueous surfaces covered with biotin-functionalized lipid monolayers, and indicated long-range orientational order of the molecules within these domaina.5 However, the limited spatial resolution of the images left open important questions regarding the microscopic organization of these systems: Where is the protein located with respect to the liquid surface, what is the f i ithickneee, and what is the lateral protein density at the interface? Electron diffraction from heavy atom stained fiis on electron microscopy grids revealed that the bound protein is in a crystalline form after transfer.6 Atomic force microscopy7and force measurementsbetween soIid/buffer interfacess have indicated that the protein forms single monolayers upon binding to f u n c t i o n h d solid substrates, and the binding was directly visualized by surface plasmon
1 Institute
(4) Green,N. M.Adv.ProteinChem.1975,29,85. Bayer,E.A,,Wilchek, M., Eds. Methods in Enzymology; Academic Press: San Diego, 1 W , VOl. 184. (5)Ahlers, M.; Blankenburg, R.; Grainger, D. W.; Meller, P. H.; Ringsdorf, H.; Salesse, C. Thin Solid Films 1989, 180, 93. (6) Dmt,S. A.;Ahlers,M.;Meller,P.H.;Kubalek,E. W.;Blankenburg, R.; Ribi, H. 0.;Ringsdorf, H.; Komberg, R. D. Biophys. J. 1991,59,387. (7) Weisenhom, A. L.; Schmitt, F.-J.;Knoll, W.; Hamma, P. K. Ultramicroscopy 1992,4244, 1125. (8)Helm, C. A.; Knoll, W.; Israelachvili,J. N. h o c . Natl. Acad. Sci. U.S.A. 1991,88,8169.
0743-746319312409-1171$04.Oo/O 0 1993 American Chemical Society
Letters
1172 Langmuir, Vol. 9,No. 5, 1993
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Figure 2. Fresnel normalized X-ray (a, b) and neutron (c, d) reflectivities of protein/lipid surface layers (a, c, on HzO;b, d, on D20). Insets depict the scattering length density profiles (electron density for X-rays, nuclear scattering densities for neutrons), used to describe the data in a global geometric model (see text).
microscopy.9 Recently, we have measured the neutron reflectivity from such films in s i t d oand have found that the protein organizes in monomolecular layers bound to the aidbuffer interface. However, the data could not be interpreted in terms of a unique model. Here we report on corresponding X-ray reflectivity studies. The combined neutron and X-ray data seta provide a more reliable picture of the structural organization of the protein at the interface.
Experimental Section Samples were prepared on a Wilhelmy film balance3Jl in 0.5 M NaCl solutions using ultrapure water that was either Millipore filtered (HzO) or thrice distilled (D2OI.l' A biotinylated lipid, compound 1 of ref 6 (see inset in Figure 3), was spread at the interface from CHC13. SA was injected underneath the lipid to a final concentration of 20 nM. Neutron studies were performed on the liquid surface reflectometer at the cold source at Risa National Laboratory, Denmark, and X-ray studies on the D4.1 reflectometer at HASYLAB, DESY,Hamburg, Germany. Both machines have been described elsewhere.3Jl-13 (9) Schmitt, F.-J.; Blankenburg, R.; Hiussling, L.; Ringsdorf, H.; Weisenhorn, A. L.; Hansma, P. K.; Leckband, D. E.; Israelachvili, J. N.; Knoll, W. In Synthetic Microstructures in BiologicalResearch; Schnur, J. M., Peckarar, M., Eds.; Plenum Press: New York, 1992; p 147. (10) Vaknin, D.; Als-Nielsen,J.; Piepenstock, M., Lbche, M. Biophys. J. 1991, 60,1545. (11)Vaknin, D.; Kjaer, K.; Als-Nielsen, J.; LBsche, M. Biophys. J.
Results Prior to the reflectivity studies we have examined similarly prepared samples by fluorescence microscopy.l4 The micrographs show that the protein organizes qualitatively similarly but quantitatively differently on NaC1/ D2O and on NaCl/H20 subphases.1° On D20, the protein forms domains with a smaller average size than on H2O under otherwise identical conditions. This has been attributed to differences in nucleation efficiency which may be associated with different interaction energies of hydrogen bonds between the protein molecules in the two solvents.10 From reflectivity curves one can extract3J0the scattering length density (SD) profiles across the interfaces (the electron density in the case of X-rays and the nuclear scattering length density in the case of neutrons). To calculate the model reflectivities, we have used the Parratt f ~ r m a l i s m . ~ JThe ~ J ~X-ray reflectivity of a biotinylated monolayer prior to protein injection into the subphase is shown in Figure 1. The data are normalized by the reflectivities of the pure subphase (the Fresnelreflectivities RF).The inset depicts the SD profile across the interface which is used to model the data. The profile extends over a distance of about 30 A. The electron density of the lipid's tail region is lower than that of closely packed hydrocarbon chains,16consistent with a low density of the deposited film, one molecule per 120 A2. To account for the fitted electron density of the lipid head groups, which
1991,59, 1325.
(12)Vaknin,D.;Kjaer,K.; Als-Nielsen,J.;Lbche,M.Makromol. Chem. Macromol. Symp. 1991,46,383. (13) Als-Nielsen, J.; Pershan, P. Nucl. Instrum. Methods 1983,208, 545.
(14) Lbche, M.;MBhwald, H. Reu. Sci. Instrum. 1984,55, 1968. (15) Parratt, L. G. Phys. Reu. 1954, 95, 359.
(16)Helm, C. A.; Tippmann-Krayer, P.; Mahwald, H.; Als-Nielsen,J.; Kjaer, K. Biophys. J. 1991,60, 1457.
Letters
Langmuir, VoZ.9, No. 5, 1993 1173 Table I. In Situ Structure of Streptavidin Bound to a Biotinylated Monolayer at the Air-Water Interfacea
d
Figure 3. Schematic representation of the layer structure. T h e lipid monolayer is distorted by the tightly bound protein. Scattering length density profiles are shown on the left (n, neutrons; x, X-rays; H, H2O; D, D20). Outline dimensions and mutual distances are to scale.
is too large for the lateral density of the monolayer, we assume interpenetration of water and of salt. The solid line in the main figure shows the calculated reflectivity from this profile with surfaceroughness taken into account, a=33. Normalized X-ray and neutron reflectivities from SA solutions injected beneath such biotinylated lipid monolayers are shown in Figure 2. Included as solid lines are the calculated reflectivities from a global model that fits all data sets simultaneously. In modeling the data, we have assumed that the interface structure consists of three layers3of homogeneous SDs. The first and second layers incorporate hydrocarbon tails and nonbound head groups of the biotinylated lipid, respectively; see Figure 3. The third layer contains the protein, two bound biotin head groups per SA, and associated water m01ecules.l~ The independent parameters of the model are defined in geometrical terms, with appropriate packing constraints of the constituents, in a composition-space refinement18 approach. The X-ray and neutron SDs are determined by these common parameters and the respectivescattering lengths.11J2 In principle, this procedure allows simultaneous refinement of one general model structure from d l data sets under the assumption that the structure of the interface is independent of the isotopic identity of the sample. However, in view of the different macroscopic protein organizations on different subphases, we allowed for two independent average areas per protein molecule in films on H20 and D20,AprHzoand AprDzo. In addition, differencesin the surface concentrations of the lipids were allowed in the refinement to take into account slight differences in the preparation. The motivation for such a deviation from the singlemodel concept comes from nucleation theory:19 Since D20 as a solvent is more structured than H20,2Othe free energy gain associated with the solvent-mediated aggregation of protein molecules will be larger here than in regular water, possibly due to a higher interaction energy of deuteron bonds than of hydrogen bonds. A smaller average size of the macroscopic protein domains on D20 buffer than on H20may then reflect this difference in interaction energy, as the size of critical nuclei will be reduced on D20, leading to the observed reduction in domain size. Microscopically, an increasein interaction energy will result in a reduction in bond length, i.e., in protein-protein distance, and we expect AprDZo< A PrHzo. (17) It was not possible to describe the data by models in which all the nonbound lipid head groupsextended into the space between the proteins. Refinement of such models brought the scattering length of the lipid head groups into the lipid tail region, i.e., back into the configuration depicted in Figure 3. (18)Wiener, M. C.; White, S. H. Biophys. J. 1991,59, 174. (19)Adamson, A. W. Physical Chemistry of Surfaces, 5th ed.;Wiley: New York, 1990; p 364 ff, Chapter IX. (20) Reichardt, C. Solvents and Solvent Effectsin Organic Chemistry, 2nd ed.; VCH Verlagsgesellschaft: Weinheim, 1988; p 281 ff.
Independent Parameters 4900 f 1000 k2 average area per protein on H2O subphase 4300 f 1000 A2 average area per protein on D20 subphase 42f2A protein layer thickness 66 000 f 6000 A3 water excluded volume of SA in the film 120-160 A2 area per lipid molecule in different data sets 8A thickness of lipid tail layer 5 A t l3A thickness of lipid head layer 4A surface roughness 4600 f 1500 3800 f 1500 650 f 50 As 30-40
Dependent Parameters water molecules per SA (H2O subphase) water molecules per SA (D20subphase) water excluded volume of lipid head group lipid molecules in surface monolayer per protein
Scattering Length Densitiese protein layer 2blhead)lApdpr lipid head group layer =blhead/ Vlhead =blbil/Aldlbil lipid tail layer =(bSA 4-
h b , 4-
a The volume Apdpr of the protein compartment comprises one SA molecule (volume VSA),n, water molecules (V, = 30 A:
