Langmuir 1993,9, 2436-2447
2436
Bacteriorhodopsin in Langmuir-Blodgett Films Imaged with a Scanning Tunneling Microscope H. E.-M. Niemi,**tM. Ikonen,$ J. M. Levlin,? and H. Lemmetyinenj Laboratory of Physics, Helsinki University of Technology, 02150 Espoo, Finland, and Department of Chemistry, Physical Chemistry Division, P.O. Box 13 (Meritullinkatu IC), FIN-00014 University of Helsinki, Finland Received March 9,1993. I n Final Form: June 2 , 1 9 9 9
Mono- and bilayer Langmuir-Blodgett (LB) f i i s of bacteriorhodopsin (bR) in an added soya phosphatidylcholine matrix were studied with scanning tunneling microscopy (STM). Layer thicknesses of about 2.5 nm and 4.5-4.8 nm, were observed in STM images and correspond well with the length of phospholipidsand bR molecules, respectively. The different layer thicknessesobtained show the existence of mono- and multilayer structures. Molecular resolution images indicate that bR protein is embedded in the phospholipid matrix. In addition, local ordering was imaged of bR units of about 6 nm similar to native purple membrane (PM). Intermolecular distances of 0.41 f 0.03 and 0.50 f 0.03 nm, indicating methyl groups of phospholipids,and spacings of 0.70 f 0.06 and 0.65 f 0.10 nm, corresponding to the polar ends of phospholipids, were observed. Evidence of local areas of monolayers and bilayers of bare phospholipids was found. On the basis of the results obtained by STM imaging, a possible model is proposed for the structure of bRsoya-PC LB film. 1. Introduction The single protein, bacteriorhodopsin (bR), of purple membrane (PM) of Halobacterium halobium' is most commonly known as a transmembrane protein which pumps protons across the cell membrax~e.~~~ In PM, the single protein bR is arranged in a highly stable crystalline hexagonal lattice of p 3 ~ y m m e t r y . ~ In?this ~ lattice, three bR molecules are close enough to form excitonically coupled trimers. The dimensionsof the unit cell in native PM of bR with p 3 symmetry have been found to be about 6.3 nm1p4 and can be reduced to about 6.0 nm5 without losing thep3 symmetry. The different ways of crystallizing bR of PM have led to the formation of new stable twodimensional crystal forms of PM with an orthorhombic lattice having ~22121symmetry: as well as the formation of three-dimensional crystals of bRM and three-dimensional crystalline arrangementsof the original PM.9J0The possibility of constructing the membrane-like structure of bR using the Langmuir-Blodgett (LB) film deposition technique offers the opportunity of orientating the direction of the bR molecules and thus observing the structure of bR in an added soya-PC matrix. Since the invention of STM by Binnig and Rohrerl1J2 over a decade ago, STM imaging has proved to be a useful technique giving valuable results, not only from metals
* T o whom correspondence should be addressed. Helsinki University of Technology. University of Helsinki. e Abstract published in Advance ACS Abstracts, August 15,1993. (1) Oesterhelt, D.; Stoeckenius, W. Nature, New Biol. 1971,233,149. (2) Danon, A.; Stoeckenius, W. R o c . Natl. Acad. Sci. U.S.A. 1974,71, t f
1234. (3) Oesterhelt, D.; Stockenius, W. R o c . Natl. Acad. Sci. U.S.A. 1973, 70, 2853. (4) Henderson, R. J. Mol. B i d . 1976, 93, 123. (5) Henderson, R.; Jubb, J. S.; Rossman, M. G. J. Mol.Bio1. 1982,154, 501. (6) Michel, H.; Oesterhelt, D.; Henderson, R. Roc. Natl. Acad. Sci. U.S.A. 1980, 77, 338. (7) Michel! H.; Oesterhelt, D. In Methods in Enzymology; Packer, L., Ed.; Academic Press: New York, 1982; Vol. 88, Chapter 14. (8) Schertler, G. F. X.; Lozier, R.; Michel, H.; Oesterhelt, D. EMBO J. 1991, 10, 2353. (9) Henderson, R.; Shutton, D. J. Mol. Biol. 1980, 99,139. (10) Tsygannik, I. N.; Baldwin, J. M. Eur. Biophys. J. 1987, 24, 263. (11) Binnig, G.; Rohrer, H. Helv. Phys. Acta 1982, 55, 726. (12) Binnig, G.; Rohrer, H. Surf. Sci. 1983, 126, 236.
and semiconductors but also from special organic molecules benzene coadsorbed with carbon such as na~hthalene,'~ m ~ n o x i d e , ~liquid ~ J ~ crystals,l6 organic molecular crysto mention t a l ~ ,and ' ~ biological materials suchas only a few. At the moment, STM and the atomic force microscope (AF'M) are still the only methods capable of giving information directly at molecular level without averaging information over large areas, a point of especial interest in the case of LB film studies. The main advantage of the STM technique is that it can provide precise values of absolute heights of different structures.21*22 Until now, only two studies with molecular resolution of phospholipids deposited on highly oriented pyrolytic graphite (HOPG) have been r e p ~ r t s d . ~Experiments ~?~~ have also been reported where liposomes of phosphatidylcholines dried onto the substrate were imaged on a large scale with STM.25@More effort, however, has been put into investigating different proteins. The *surface protein HPI (hexagonally packed intermediate layer) of the bacterium Deinococcus radiodurans has also been imaged with STM. Both u n c ~ a t e and d~~ metal-coated ~~~ ~~
~13~Hallmark,V.M.;Chiang,S.;W~ll,Ch.J.Vac.Sci.B1991,9,1111. (14) Ohtani, H.; Wilson, R. J.; Chiang,S.; Mate,C. M. Phya. Rev. Lett. 1988,60, 2398. (15) Chiang,S.; Wileon, R. J.; Mate,C. M.; Ohtani, H. J. Microsc. 1988, 152, 567. (16) Spong, J. K.; Mizes, H. A.; LaComb, L. J., Jr.; Dovek, M. M.; Frommer, J. E.; Foster, J. S. Nature 1989,338, 137. (17) Sleator, T.; Tycko, R. Phys. Rev. Lett. 1988,60,1418. (18) Driacoll,R. J.;Youngquist, M. G.; Baldeswieler,J. D.Nature 1990, 346, 294. (19) M. Salmeron, M.; Beebe, T.;Odriozola,J.; Wilson, T.; Ogletree, D. F.; Siekhaus, W. J. Vac. Sci. Technol. A 1990,8, 636. (20) Dunlap, D. D.; Bustamante, C. Nature 1989,342, 204. Guckenberger,R. Roc. (21) Wang, Z.; Hartmann, T.; Baumeister, W.; Natl. Acad. Sci. U.S.A. 1990,87,9343. (22) Fisher, K. A.; Whitfield, S.L.; Thomson, R. E.; Yanagimoto, K. C.; Gustafsson, M. G. L.; Clarke, J. Biochim. Biophys. Acta 1990,1023, 325. (23) Hbrber, J. K. H.; Lang, C. A.; Hirnsch, T. W.; Heckl, W. M.; Mbhwald, H. Chem. Phys. Lett. 1988,145,151. (24)Lang, C. A.; Hbrber, J. K. H.; Hirnsch, T. W.; Heckl, W. M.; Mbhwald, H. J. Vac. Sci. Technol. A 1988,6,368.
(25)Luo,C.;Zhu,C.;Ruan,L.;Huang,G.;Daj,C.;Cheng,Z.;Bai,C.; Su, Y.; Xu, S.; Lin, K.; Baldeswieler, J. D. J. Vac. Sci. Technol. A 1990, 8,684. (26) Su, Y.-X.; Jiao, Y.-K.; Xu, S.-D.; Yao, J.-E.; Lin,K.-C. J. Vac. Sci. Technol. A 1990,8,695.
0743-746319312409-2436$04.O0/0 0 1993 American Chemical Society
Bacteriorhodopsin in LB Films
(Pt-Ir-C)% HPI samples have revealed layered and regularly ordered structures on a nanometer scale. Individual protein moleculeshave also been imaged.30*31 The STM study of periodical partical arrangement of crystalline lysozyme32 is one example of imaging protein crystals. HBrber et al.33have demonstrated the use of STM in studying natural PM in a natural environment and of isolated protein molecules deposited on graphite, showing in their work the capability of STM to image surface cell membranes. Guckenberger et al.34also report studies of uncoated PM, although successful imaging depended on the voltage and current used. Golubok et al.35have imaged vesicles obtained from native purple bacteria Rhodospirillum rubrum. Only one study of a protein-incorporated LB film has been reported, namely a study of glucose oxidase-incorporating arachidic acid LB film using AFM.36 In this work a scanning tunneling microscope (STM) operating at ambient air pressure is used for studying the surface structure of the protein bR of PM in an added soya-PCmatrix LB filmin order to obtain new information about the structure. STM is applied for studying different types of monolayers and bilayers of bRsoya-PC LB film deposited on HOPG. The STM images give information about the homogenity of the LB film and reveal the layer structure. Molecularresolution images have been obtained from phospholipid headgroups in these films and from the bR protein embedded in phospholipid matrix. Also locally ordered areas of bR are shown. On the basis of these results we propose a model for layer structures arising from the deposition of this LB film. 2. Experimental Section 2.1. LB Film Deposition. The purple membrane (PM) of
bacteriorhodopsin (bR) was supplied by Professor Mitsner (Institute of Fine Chemical Technology of Moscow) and L-aphosphatidylcholinefrom soya beans (soya-PC) was obtained from Sigma Corporation. Both substances were used without further purification. The Langmuir-Blodgett (LB) films of bRsoya-PC were prepared using a KSV 5000 film depositionsystem. The hexane suspensionsof bRsoya-PC at a weight ratio of 7:l were prepared from cold solutions of PM in basal salt (CbR = 0.49 mg/mL) and soya-PC in hexane (c = 0.28 mg/mL) as described The cold hexane suspension was spread under atmospheric conditionson a water subphase (pH = 6.5-7.0 and t = 19.5 f 0.2 "C) to a surface pressure of at least 1.7 mN/m. It was essential to allow the spread film to stabilize.38*39The Langmuir film of bRsoya-PC was compressed to a deposition pressureof 40 mN/m with a barrier rate of 15mm/min. The dipping rates for vertical (27) Guckenberger, R.; Wiegrgbe, W.; Hillebrand, A.; Hartmann, T.; Wang, Z.; Baumeister, W. Ultramicroscopy 1989,31, 327. (28) Schabert, F.; Hefti, A.; Goldie, K.; Stemmer, A.; Engel, A.; Meyer, E.; Overney, R.; Gbtherodt, H.-J. Ultramicroscopy 1992,42-44,1118. (29) Amrein, M.; Wang, Z.; Guckenberger, R. J . Vac. Sci. Technol. B 1991, 9, 1276. (30) Wells, T. N. C.; Stedman, M.; Leatherbarrow, R. J. Ultramicroscopy 1992,42-44,1200. (31)Thompson, N. H.; Miles, M. J.; Tatham, A. S.; Shewry, P. R. Ultramicroscopy 1992,42+, 1204. (32) Littke, W.; Haber, M.; Gbtherodt, H.-J. J. Cryst. Growth 1992, 122,80. (33) H&ber, J. K. H.; Schuler, F. M.; Witzemann, V.; Schr6ter, K. H.; Mtiller, H.; Ruppemberg, J. P. J. Vac. Sci. Technol. B 1991,9,1214. (34)Guckenberger, R.;Hacker, B.; Hartmann,T.; Scheybani,T.; Wang, Z.; Wiegrlbe, W.; Baumeister, W. J. Vac. Sci. Technol. B 1991,9,1227. (35) Golubok, A. 0.; Vinogradova, S. A.; Tipisev, S. Y.; Borisov, A. Y.; Taisova, A. S.; Kolomytkin, 0. V. Ultramicroscopy 1992,42-44, 1228. (36) Fujiwara, I.; Ohnishi, M.; Seto, J. Langmuir 1992,8, 2219. (37) Hwang, S.-B.; Korenbrot, J. I.; Stoeckenius, W. J . Membr. Biol. 1977,36,115. (38) Ikonen, M.; Peltonen, J.; Vuorimaa, E.; Lemmetyinen, H. Thin Solid Films 1992,213, 277. (39) Ikonen, M.; Sharonov, A.; Tkachenko, N.; Lemmetyinen, H. Accepted for publication in Ad. Mat. Opt. Electr.
Langmuir, Vol. 9, No.9, 1993 2437
Figure 1. A schematic presentation of the structure of the tunneling unit. The sample holder is attached to a coarse adjustment differential microscrew table and the tip is fixed by friction to the short z-piezo. The scanner consists of four piezo tubes connected to a metal cube, two piezos being in the z-direction. The tip is brought to tunneling distance with the long z-piezo, which is also used for compensating thermal drift. The short z-piezo is used for following the surface topography during scanning. The conventional stack of steel plates is used for secondary vibration isolation. depositionwere 15 mm/min upward and downward. HOPG was used as a substrate for bRsoya-PC films. A clean surface of HOPG was achievedby cleaving it with adhesivetape just before use. The plane of the HOPG substrate was parallelto the barrier in order to reduce flow gradients during the deposition."' For comparison,some samples were deposited on a HOPG Substrate perpendicular to the barrier. Different Types of Monolayers and Bilayers. The monolayer bRsoya-PC sampleswere depositedeither from up to down (1)asan x-type LB film, in order to orient the extracellular surface of bR39*41*42 toward the HOPG substrate and the cytoplasmic surface toward air, or from down to up (t) as a z-type LB film, in order to orient the cytoplasmic surface toward the HOPG substrate and extracellular surface toward the air. The bilayer bRsoya-PC films were deposited as x- or z-type bilayer films or asy-type bilayer films. They-type film is obtained by depositing the first layer either as an x- or z-type film, which is then covered with a film of the other type. In the y-type bilayer films, the cytoplasmic surfaces of bR proteins are facing each other when the deposition is started as an x-type film and continued as a z-type film. Thus, the extracellular surface of the first layer is toward HOPG and the extracellular surface of the second layer is toward the air. The other possibility is to deposit in the opposite way so that the extracellular surfaces are facing each other. 2.2. STM Construction. Mechanics. The experimentsin this study were performed with a STM operating at ambient air pressure. We have constructed a scanningtunneling microscope following the basic principles of the pocket size STM designed by Gerber et al.43 The construction was designed for studying biological and organic macromolecules. The configuration of the tunneling unit is shown in Figure 1. The sample holder is attached to a differential microscrew translation table which is used for coarse approaching. The XYZ scanner consists of four PZT-typepiem tubes. Two of them are used for scanningsurfaces in the x- and y-direction. The long z-piezo is used for final approaching and also for compensating thermal drift during tunneling, while the short z-piezo is used for fast movements of the STM tip in following the surfacetopography duringscanning. The tunneling unit is mounted on a stack of steel plates and is (40) Hann, R. A. In Langmuir-Blodgett Films;Roberta,G.,Ed.; Plenum Press: New York, 1990; Chapter 2. (41) Rawn, J. D. In Biochemistry, International Edition;Neil Pattereon Publishers, Carolina Biological Supply Company: Burlington, NC, 1989; p 1032. (42) Lin, G. C.; Awad, E. S.;El-Sayed, M. A. J. Phys. Chem. 1991,95, 10442. (43) Gerber, Ch.; Binnig, G.; Fucha, H.; Marti, 0.;Rohrer, H. Rev. Sci. Instrum. 1986,57,221.
Niemi et al.
2438 Langmuir, Vol. 9, No. 9, 1993 situated inside a chamber made of aluminum. The dynamic scanning area with low voltages is up to 375 nm X 375 nm and with high voltages up to 1.5 Hm X 1.5 Hm. Electronics and Software. The feedback electronics are of the conventional type. The bias voltage is applied to the tip, the sample being grounded through a current amplifier. The output from the current amplifier is fed through an absolute value amplifier and a logarithmic amplifier to an analog feedback integrator. The integrator drives either the low voltage amplifiers (for atomic resolution images) or the high voltage amplifiers (for large scan areas) controlling the z-piezo. The x-sweep voltage is generated with electronics, while the y-sweep is controlled by software with a 12-bit D/A converter. Data are collected with a 12-bit A/D converter from the feedback integrator output (constantcurrent mode) or fromthe logarithmicamplifier output (constant height mode). Z/V measurements can be made by interrupting the feedback loop with a sample-and-hold circuit and sweeping the bias voltage, while collecting data from the logarithmic amplifier output. A Sperry PC-AT microcomputer with home-written control software is used for STM control and data acquisition. Images are analyzed in a separate Macintosh I1 microcomputer with a commercial image analysis package. 2.3. STM Measurements. Calibration. Atomic images of HOPG were used for calibrating the piezo system in the x- and y-directions. Values in the z-direction were confiied with the atomic steps of graphite as well as with images of the atomic steps of gold evaporated on mica. Conditions. STM images were obtained at ambient air pressure at room temperature, of about 20-25 "C. Wet filter paper was placed in the measurement chamber and the system was allowed to stabilize before measuring. Relative humidities of about 70-8i5 % measured with a capacitancegaugewere present most of the time. In addition, measurementsin ambient humidity were also carried out. Only mechanically cut Pt/Ir (80%/20%, diameter 0.5 mm) tips were employed. STM images were taken in the constant current topographic mode. Some of the atomic resolution images were obtained using the constant height mode. A large variety of tip bias voltages, ranging from small voltages of the order of & 50 mV up to large voltages of the order f 9.5 V with the sample grounded, were used in the measurements. The tunneling current was usually varied between 0.05 and 2 nA. These parameters correspond to tunneling resistant values of 0.1-180 GR. Image acquisition times depended on image size. These times used were between 5 and 250 s corresponding to scanning speeds of 50 to 1 lines/s. Most of the STM images presented here are raw data with a few exceptions, where Fourier filtering has been applied.
3. Results HOPG has been widely used as a substrate for STM studies of organic molecules, biological objects, and LB films. It is well-known that besides the large atomically flat areas, other features also occur. For example, there have been observations, made by us as well, of a number of different morphologicalstructures such as cleavage steps of different heights, ridges, strands, flakes, and even graphite f i b e r ~ . l ~Images * ~ of HOPG also show superstructures4- and Moire patterns.51 However, the major advantage of HOPG is that the flat, clean surface is easily achieved by cleaving it in air. In addition, the surface is rather inert. The different morphological structures on HOPG mentioned above pose problems in the interpre(44)Chaug, H.; Bard, A. J. Langmuir 1991, 7,1143. Chaug, H.; Bard, A. J. Langmuir 1991, 7, 1138. (45) Liu, C.-Y.; (46) Kuwabara, M.; Clarke, D. R.; Smith, D. A. Appl. .. Phys. Lett. 1990, 56,2396. (47) Sawamura, M.; Womelsdorf, J. F.; Ermler, W. C. J. Phys. Chem. 1991,95,8823. (48) Buckley, J. E.; Wragg, J. L.; White, H. W. J. Vac. Sci. Technol. E 1991,9, 1079. (49) Xhie, J.; Sattler, K.;MUer, U.; Venkateswaran, N.; Raina, G. Phys. Rev. E 1991,43,8917. (50) Shedd, M.; Russell, P. E. Surf. Sci. 1992, 266, 259. (51) Albrecht, T. R.; Mizes, H. A.; Nogami, J.; Park, S.-I.; Quate, C. F. Appl. Phys. Lett. 1988, 52, 362.
tation of images of LB films on HOPG. Even so it seemed to be the best choice for the studies of bRsoya-PC LB film. We have tried to avoid drawing any conclusions from rectilinear edge structures and even round-shaped edge structureswhich might arise from HOPG, unless we could prove beyond doubt that there is definitely a LB layer. This was established by either taking a molecular remlution image or making a surface roughness comparison, and further by correlating layer thicknesses. 3.1. Homogenity of the bRSoya-PCLB Films. The first samples in this study were prepared by vertical dipping with the substrate perpendicular to the barrier. This can induce flow gradients on the substrate and result in incomplete coverage. Therefore in later samples, deposition was done with the substrate parallel to the barrier in order to reduce flow gradients during the deposition.a T w o different types of monolayers were deposited either in the down to up direction, referred to as (!)-type, or in the up to down direction, referred to as (4)-type. The bilayers were deposited either in the same directions, referred to as (ll>-t e and (tt>-t different directions, referred to as ( ?)-typeand (t )-type. Or According to our STM measurements, the surface structures of bRsoya-PC LB films were not homogenous throughout the samples, but wide areas of homogenity were imaged. LB film boundaries of different heights were found and they had in common round-shaped edges. Occasionally holes and bare graphite were observed. In a few images, the LB film material seemed to be fixed only on the edges of graphite flakes and steps. Some LB films showed different types of crystal forms and aggregations. The Layer Structure of Films Deposited as Monolayers. Monolayers of the (ll-type deposited on HOPG parallelto the barrier showed quite homogenousfilms with small holes and protrusions rising from the film. The existence of holes in DPPC (dipalmitoyl phosphatidylstudied by STM as well as in fatty acid LB filmss3 in AFM studies have been reported. A notable finding in our study is that the shape and size of the holes in the monolayer film did not change noticeably during repeated scanningin contrast to the changes often observed in AFM measurements, sometimes due to force interactions." Closer examination revealed that the film was not a monolayer, but consisted of layers of varying heights changingfrom region to region, but the same height covered some tens or hundreds of nanometers. The existence of edges in these large areas allowed us to estimate height differences between levels. The holes in the LB film could also be utilized in height determinations. Figure 2a shows an image of a 34 nm X 34 nm area with changing heights taken from a sample covered with a monolayer of the (Jl-type bRsoya-PC LB film. For the most part, smooth areas imaged in this region seemed to be formed of a film with an average layer thickness of 2.6 f 0.3 nm as can be seen from cross section A in Figure 2b. The heights of the protrusions in these areas varied from 4.5 nm to 4.8 nm, which is 2.0 nm to 2.3 nm higher than the average layer thickness, &B can be noticed from cross section B shown in Figure 2c. The thickness of a phospholipid Langmuir film layer is 2.2-2.4 nm at a surface pressure of over 20 mN/m.55 The layer thickness of 2.5 & 0.3 nm observed in the present studyis in good agreement
YP
p.'
(52) Eng,L.; Hidber, H.-R.;Rosenthaler, L.; Staufer, U.; Wiesendanger, R.; Gbtherodt, H.-J.; Tamm,L. Vac. Sci. Technol. A 1988,6, 358. (53) Fuchs, H.; Chi, L. F.; Eng, L. M.; Graf, K. Thin Solid F i l m 1992, 2101211, 656. (54) Leung, M.; Goh, M. C. Science 1992,265,6166. (55) Duchanne, D.; Salewe, C.; Leblanc, R. M. Thin Solid F i l m 1986, 132, 83.
Langmuir, Vol. 9, No. 9, 1993 2439
Bacteriorhodopsin in LB Films
60-
20 nm
nm 14
SO -
8
40 -
30 -
7
4
0 20
IO
0 nm
70
a)
- 3
nm 10
5
nm
b)
-
nm
nm
IS
-
=,---7
-~
5
-r
-
10
2s
30
? .'
~
-
20
IS
I *
20
25
I0
20
30
40
SO
Tv-L 60
70
80
1
61 90
b)
Figure 3. (a) STM image of a monolayer of (?)-type bRsoyaPC LB film deposited on HOPG. Different layer thicknesses can be observed. The size of the image is 107 nm X 107 nm. Tunnelingcurrent of 0.2 nA and bias voltage of -2.5 V were used. (b) Cross section (raw data) from part a presenting a layer thickness of about 4.8 nm and a layer thickness of about 2.5 nm.
