Imaging Globular and Filamentous Proteins in Physiological Buffer

3529. Imaging Globular and Filamentous Proteins in. Physiological Buffer Solutions with Tapping Mode Atomic. Force Microscopy. Monika FritzY*9t9$ Manf...
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Langmuir 1995,11,3529-3535

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Imaging Globular and Filamentous Proteins in Physiological Buffer Solutions with Tapping Mode Atomic Force Microscopy Monika FritzY*9t9$Manfred Radmacher,? Jason P. Cleveland,t Miriam W. Allersma,g Russell J. Stewart," Ralph Gieselmann,lPv Paul Janmey,l Christoph F. Schmidt,$ and Paul K. Hansmat Physics Department, Marine Science Institute, University of California, Santa Barbara, California 93106,Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109,Bioengineering Department, University of Utah, Salt Lake City, Utah 84112,and Harvard Medical School, Division of Experimental Medicine, Brigham and Women's Hospital, LMRC 301,221 Longwood Avenue, Boston, Massachusetts 02115 Received March 27, 1995. I n Final Form: May 30, 1995@ Two differenttypes ofproteinswere imaged in buffer solution with tapping mode atomic force microscopy (AF'M) in liquids: the globular proteins lysozymeand monomeric actin (G-actin);the filamentousproteins actin (F-actin)and microtubules. To calibrate the AFM in contact and tapping modes in liquids, a sample was prepared with features that are comparable to the height of a single protein molecule: steps in mica with a height of 1 nm. Single globular molecules of lysozyme and G-actin could be readily imaged in physiological buffer at pH 7 and lower, whereas F-actin could only be imaged stably without any visible damage at around pH 6. The helical pitch of the actin filaments was measured to be 37 nm, which is in good agreement with data from X-ray diffraction and transmission electron microscopy (TEM). The negatively charged microtubules could not be imaged on freshly cleaved mica; instead, a method was established to adsorb them to silanized glass. Both protein types could be imaged stably with loading forces of about 200 pN. The height of the proteins was larger than the expected height measured by X-ray diffraction on protein crystals. Mechanical properties and/or electrostaticinteractions may contribute to the image formation. Further work is needed to understand the height measured by tapping mode in liquids. We show here that single, globular protein molecules and protein filaments can be imaged easily and stably in buffer solution.

Introduction AF'M has proven to be a very useful tool for biological applications. Soon after its invention' it became possible to image in an aqueous e n v i r ~ n m e n t .Whole ~ , ~ living cells were imaged during the performance of their biological function with a resolution of about 50 nm.4-6 Recently, force measurements were performed between individual biological molecules,namely avidin and biotin.'a Imaging of single protein molecules by AF'M is oRen complicated by high lateral and loading forces, and impressive results are rare.9 It has been shown that covalently binding protein molecules to solid substrates is one possible

* Author to whom correspondence should be addressed at Physik Department der TU Munchen, Institut fur Biophysik, E22, James Franck Strasse, 85748 Garching, Germany. Telephone: 089-3209 2471. FAX. 089-3209 2469. E-mail: [email protected]. Physics Department, University of California. Marine Science Institute, University of California. 8 University of Michigan. 11 University of Utah. Brigham and Women's Hospital. vRalph Gieselmann died on June 11, 1994. This paper is dedicated to him. * Abstract published inAdvance ACSAbstracts, August 1,1995. (1) Binnig, G.;Quate, C. F.;Gerber, C. Phys. Rev. Lett. 1986,56,930. (2) Weisenhorn,A. L.; Hansma, P. K.; Albrecht, T. R.; Quate, C. F. Appl. Phys. Lett. 1989, 54, 2651. (3) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Albrecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. K. Science 1989, 243, 1586. (4)Fritz, M.; Radmacher, M.; Gaub, H. E. Biophys. J.1994,66,1328. (5) HBberle, W.; Horber, J. K. H.; Ohnesorge, F.; Smith, D. P. E.; Binnig, G. Ultramicroscopy 1992,42-44, 1161. ( 6 )Hoh, J. H.; Schoenenberger,C.-A.Biophys. J. 1994,107, 1105. (7) Florin, E.-L.;Moy, V. T.; Gaub, H. E. Science 1994,264, 415. (8)Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994,10,354. (9) Yang, J.; Mou, J.; Shao, Z. Biochim. Biophys. Acta 1994, 1199, 105.

approachlo to reproducibly image proteins. Another approach involvesthe use of a new imaging mode: tapping in liquids,ll where the probe is gently oscillating and "taps" the sample. In this mode, single biologicalmolecules can be investigated with high resolution. Most importantly, it is even possibleto observethe motion of single molecules; DNA strands were imaged during their degradation by an enzyme,12 and lysozyme was observed during its enzymatic activity.13 Reasons for the more gentle imaging of soft samples in tapping mode include the lack of lateral forces that push aside weakly adsorbed molecules and smaller loading forces (see Figure 1). Typically, when the AFM tip approaches the sample, the tip jumps to the sample at a critical distance due to van der Waals attraction. This mechanical instability sets a lower limit to the imaging force that can be used. This instability is circumvented in tapping mode, and therefore, the only limit of the loading force is the accuracy of the electronics and the detection system. Another important point is that in contact mode the loading forces vary because of thermally induced drift of the ~anti1ever.l~ This effect is not present in tapping mode; the ac detection methods used eliminate this problem. Thus, soft samples can be imaged at very low forces. There is no direct way of (10) Karrasch, S.; Hegerl, R.; Hoh, J. H.; Baumeister,W.;Engel, A. PNAS i s s 4 , 9 i , a36. (11) Hansma,P. K.; Cleveland,J. P.;Radmacher,M.; Walters,D.A.; Hillner, P. E.; Bezanilla, M.; Fritz, M.; Vie, D.; Hansma, H. G.;Prater, C. B.; Massie, J.; Fukunaga, L.; Gurley, J.; Elings, V. Appl. Phys. Lett. 1994, 64, 1738. (12) Bezanilla, M.; Drake, B.; Nudler, E.; Kashlev, M.; Hansma, P. K.; Hansma, H. G. Biophys. J. 1994, 67, 1. (13) Radmacher,M.; Fritz, M.; Hansma, H. G.;Hansma, P. K. Science 1994,265, 1577. (14)Radmacher, M.; Cleveland, J. P.; Hansma, P. K. Scanning, in press.

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Figure 1. Schematic drawing of an atomic force microscope operating in (A) tapping mode in liquids and (B)contact mode. In tapping mode in liquids (A) the cantilever is oscillated sinusoidallywith a frequency between 15 and 35 kHz.Lateral forces are minimized and the loading force can be controlled very precisely. In contact mode (B)soft samples are easily distorted by lateral forces. calibrating the loading forces in tapping mode, which can be easily achieved in contact mode. From the elastic indentation of soft molecules we estimated that the effective loading forces in our case are below 200 pN.15913 Here, we report on further investigations using tapping mode in liquid on the globular proteins lysozyme and G-actin (globular actin) and on the filamentous proteins F-actin (filamentous actin) and microtubules. Those proteins could be imaged easily and reproducibly. Lysozyme is a small, well-studied enzyme with a molecular weight of about 17 kDa and a size of 3.5 x 3.5 x 5 nm as measured by X-ray diffraction on protein crystals.16 It is a single chain molecule with a negatively charged active cleft and an overall positive surface charge which results in a PI (isoelectric point) of 11.1. It can be easily adsorbed to negatively charged surfaces like mica and does not lose its enzymatic activity in the adsorbed state.17 Recently, it has been shown that the activity of lysozyme can be observed directly with AFM.13 Its biologicalfunction is to hydrolyze bacterial cell walls and thus act as an antibacterial agent. It can be found in a variety ofvertebrate cell types like spleen and kidney and in cell secretions like milk and tears (for an overview see ref 18). Actin is found in almost all eukaryotic cells, from yeast cells to primate cells, as a major constituent of the cytoskeleton (for a review see ref 19). It has various

functions in the cell including roles in muscle contraction, cell motility, and stabilization of cell structure by linkage of the plasma membrane to the cytoskeleton. Globular, monomeric actin has a molecular weight of 42 kDa and is about 5.5 nm in diameter. Its three-dimensional structure has been determined by X-ray crystallography a few years ago.2o In the presence of 150 mM KCl andor 2 mM MgC12 monomeric actin polymerizes to filaments until an equilibrium concentration of monomers of about 1 pM is reached. The resulting filaments are about 7 nm in diameter and show a two-strand helix with a helixpitch of about 36 Although the filaments are negatively charged at neutral pH, they can be adsorbed to a negatively charged mica cleavage plane. There may be a counterion layer around the actin filament which mediates the attachment of the filaments to mica (personal communication with Jay Tang). Microtubules, another major type of filamentous polymer in eukaryotic cells, are involved in numerous cellular functions such as ciliary and flagellar motions, chromosome movement in mitosis and meiosis, structural support of cells, and intracellular transport. Microtubules are hollow tubular structures of about 30 nm in diameter; they can be up to several millimeters in length. They consist-despite the diversity of their functions-of two discrete globular, 54 kDa subunits: a-and#?-tubulin.The a- and #?-subunitsbind head to tail to each other to form protofilaments. These protofilaments are bundled together; between 8 and 15 (depending on buffer conditions) can form the microtubule cylinder. In the cylinder the rows oftubulin molecules form a slope of about 60"relative to the cylinder axis. (for overviews see refs 22 and 23). Microtubules can be assembled in vitro in the presence of 1 mM GTP, 2 mM magnesium, and 0.1 mM EGTA if the tubulin concentration is higher than 0.2 mg/mL (critical concentrati~n).~~ The filaments have excess negative charge at the physiologicalpH 7;25and, therefore, require a positively charged surface to be adsorbed for AFM imaging. We show here that an aminosilane can be used to adsorb microtubules with sufficient strength so that they can be imaged in physiological buffer solution by AFM operated in tapping mode. We report here the conditions in which the different protein molecules could be imaged and the limits of the resolution achieved on these soft samples.

Materials and Methods Substrate Treatment. Mica. Proteins were adsorbed on freshly cleaved mica. For the height calibration the freshly cleaved surface of muscovite mica was scratched with 100 nm silica spheres (Monosphere1000,Merck, Darmstadt, Germany) dissolved in deionized water (Milli Q, Eugene, Oregon) to a concentration of 1 mg/mL. Two pieces of mica were rubbed against each other with the spheresin between to create scratches down to one cleavage plane (1 nm in thickness). Glass. Glass cover slips (Electron Microscopy Sciences, Fort Washington, Pennsylvania), 15 mm in diameter, were cleaned thoroughly before coating with silane. First they were sonicated for 5 min in a saturated solution of KOH in ethanol, and then they were rinsed with deionizedwater (MilliQ,Millopore System, Eugene, Oregon) and ultrasonicated 3 times in deionized water for 3 min. They were silanized with DETA (trimethoxysilyl-

(20)Kabsch, W.; Mannherz, G.; Suck, D.; Pai, E. F.; Holmes, K. C. Nature 1990,347,37. (16)Blake,C.C.F.;Koenig,D.F.;Mair,G.A.;North,A.C.T.;Phillips,(21)Holmes,C. H.; Popp, D.; Gebhard, W.; Kabsch, W. Nature 1990, 347,44. D.C.; Sarma, V. R. Nature 1965,206,757. (22)Gelfand, V.I.; Bershadsky, A. D. Annu. Rev. Cell Biol. 1991,7, (17)Golander, C.G.;Hlady, V.; Caldwell, K.; Andrade, J. D. Colloids 93. and SuTf: 1990,50,113. (23)Hyams, J. S.;Lloyd, C. W. Microtubules;Wiley-Liss: New York, (18)Osserman, E. F.; Canfield, R. E.; Beychok, S. In Lysozyme; 1994. Osserman, E. F., Canfield, R. E., Beychok, S., Eds.; Academic Press: (24)Weisenberg, R. C.Science 1972,177,1104. New York, 1974. (25)Little, M.; Seehaus, T. Comp. Biochem. Physiol. 1988,90B,655. (19)Pollard, T.D. Curr. Opin. Cell Biol. 1990,2,33. (15) Radmacher, M.; F'ritz, M.; Cleveland, J. P.; Walters, D. R.; Hansma, P. K. Langmuir 1994,10, 3809.

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Figure 2. Mica surface scratched with glass spheres imaged with an atomic force microscope in deionized water in (A) contact mode and (B) tapping mode in liquids. The same area is imaged in the two modes. The steps are clearly visible, and they show in both modes steps of 1nm within experimental error.

propyldiethylenetriamine,United Chemical Technologies, Bristol, Pennsylvania). A sample of 1%DETA was hydrolyzed in 1 mM acetic acid for 5 min. The cover slips were immersed in this solution for 2 min, rinsed in deionized water, and sonicated for 2 min. Finally, they were dried and cured for 15 min at 150 "C. Protein Treatment and Immobilization. Lysozyme. Hen egg white lysozyme (Grade I, Sigma, St. Louis, Missouri) was adsorbed on freshly cleaved mica. A solution of 1pg/mL (-0.1 pM)lysozyme in deionized water (Milli Q, Eugene, Oregon) was incubated for 30 min directly on the mica in the fluid cell of a commercial microscope (Nanoscope 111, Digital Instruments, Santa Barbara, CA). Then the fluid was exchanged against a buffer containing 5 mM KH2P04 at pH 6 without lysozyme. For contact mode imaging lysozyme was dissolved in 5 mM KH2P04 at pH 4 and treated otherwise as above. The molecules in Figure 3B have been adsorbed to a mica surfacewhich has been scratched with glass spheres in order to create steps on the surface. This was done to have an internal calibration. The line plot was done on molecules which belong to the same step. Actin. G-actin (globular actin) from rabbit skeletal muscle was prepared by Ralph Gieselmann. It was sent on dry ice in G-buffer (2 mM Tris/HCl, pH 7.5, 0.5 mM ATP, 0.2 mM CaC12, 0.2 mM dithiothreitol, and 0.02% NaN3) and kept at -80 "C. Before the experiments 1mL of a 2 mg/mL solution of globular actin was dialyzed against three changes of 500 mL of G-buffer to load the G-actin with ATP. The freshly dialyzed protein solution was centrifuged at 100 000 g (Beckmann ultracentrifuge, Model Optima TL; Rotor, TLA 100)for 1h to remove aggregates and contaminants. Actin was polymerized in F-buffer (2 mM TrisBCl, pH 7.5, 0.5 mM ATP, 0.2 mM CaC12, 0.2 mM dithiothreitol, 0.1 M KC1,2 mM MgCl2, and 0.02% NaN3) for 12

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h at 4 "C and then stabilized with phalloidin (Sigma, St. Louis, Missouri) at a molar ratio of actin:phalloidin of 1:l for 12 h at 4 0C.26927 A 50 pL sample of the filament solution was incubated on mica using a cut pipette tip to reduce damage of the filaments. After 30 s the filaments that had not adsorbed were removed by flushingthe sample with the F-buffer, pH 6. For imagingG-actin 0.1 mg/mL G-actin in G-buffer at pH 7 was adsorbed to freshly cleaved mica for 10 min, rinsed vigorously in G-buffer, and mounted in the fluid cell. The samples were never allowed to dry. Microtubules. Microtubules were purified from bovine brain by the method of M. D. Weingarten28and stored at -70 "C until they were used. A 1.2mg/mL sample of tubulin was polymerized in PEM 50 buffer (50 mM PIPES (piperazine-NF-bis(2-ethanesulfonic acid), 1mM EGTA, 2 mM MgCl2, 2 mM NaN3, pH 6.8 with 10 pM taxol) at 37 "C for 10 min. The tubulin solution was allowed to polymerize for an additional 12h at room temperature after it was removed from the water bath. The polymer solution was centrifuged for 30 min at 14 000 rpm (Eppendorf', 5415 C, table-top centrifuge) to remove unpolymerized tubulin. The supernatant was discarded and replaced by the same volume of pure PEM 50 buffer pH 6.8 and 3 pM taxol and resuspended. A 10 pL sample of this solution was adsorbed for 10 s on DETAcoated cover slips and rinsed vigorously with PEM 50 buffer containing 3pM taxol. The sample was mounted in the fluid cell and was never allowed to dry. (26)Le Bihan, T.;Gicquaud, C. Biochem. Biophys. Res. Commun. 1991,181,542. (27)Sampath, P.;Pollard, T. Biochemistry 1980,30,1973. (28)Weingarten, M.D.;Suter, M. M.; Littman, D. R.; Kirschner, M. W.Biochemistry 1974,13,5529.

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Figure 3. Lysozyme adsorbed to mica imaged with a n atomic force microscope operated in contact mode (A) and in tapping mode in liquids (B).For contact mode imaging (A),the lysozyme buffer had to be at pH 4 or below to make the protein sufficiently positively charged to adsorb strongly. At pH higher than 5 it was not possible to image in contact mode. In tapping mode in liquids, lysozyme (B) could be imaged up to pH 8. The image in (B) is taken at pH 7. Note that the line plots show a n apparent height for lysozyme imaged in tapping mode that is larger than that in contact mode.

Instrumentation. AFM was performed with a modified version of a commercial microscope (Nanoscope 111, Digital Instruments, Santa Barbara, CAI, which could be operated in tapping mode under 1iquids.ll In this mode less damage occurs to soR samples. The cantilever is modulated sinusoidally at high frequencies. We chose one frequency between 16 and 35 kHz with an amplitude of about 5 nm (see Figure 1). The cantilever response depends on the distance between tip and sample. The cantilever response is used as the input to a feedback loop, adjusting the piezo height such that the vibration amplitude stays constant. This mode is analogous to the constant deflection mode in conventional AF'M. Various types of cantilevers were used: ShN4tips (200pm long and 12pmwide, a specialfabrication of Mark Wendman, Digital Instruments, Santa Barbara, CAI for the lysozyme images and some actin filament images; silicon tips (180 pm long, 18 pm wide, Ultralevers, Park Scientific Instruments, Sunnyvale, CA) for some actin filament images and the microtubules. Both cantilever types have a nominal spring constant of 32 mN/m. Prior to the experiments, tips were checked for the strength of adhesion forces. Only tips showing very low adhesion ( c0.5 nN) were used. Low adhesion correlated with good atomic resolution on mica and with good resolution on the adsorbed proteins and vice versa. Therefore, we believe that tips with high adhesion are either tips with a large radius of curvature at the end or, more likely, contaminated tips.

Results and Discussion Imaging small, soR molecules like proteins with high resolution in tapping mode in liquids is easily achieved because of the lack of lateral forces and the fact that the loading force can be controlled very accurately (Figure 1). In tapping mode in liquids the feedback signal which controls the tip-sample separation is the decrease of the tapping amplitude that occurs 'during approach of a surface. This decreased amplitude (set point)is translated into a height signal by the feedback loop which corrects the piezo to maintain a constant amplitude. To measure the height variation of a sample in the range of nanometers, the piezo ceramic should be calibrated with a sample which has known height features in the range of nanometers. For protein molecules of a size of about 5 nm we chose to calibrate the instrument with steps on mica that are 1 nm in height.29 Calibration of the %-Signal.The z-signal was calibrated by steps in the cleavage plane of mica. Freshly cleaved mica was scratched with small glass spheres (see Materials and Methods section). Two pieces of mica with (29) Okusa, H.; Kurihara, K.; Kunitake, T. Langmuir 1994,10,3577.

Imaging Globular and Filamentous Proteins

an aqueous solution of the glass spheres between them were slid against each other several times. After rinsing them several times, to remove mica flakes and the glass spheres, they were mounted in the atomicforcemicroscope and imaged in pure water. The z-signal of the piezo was calibrated according to the step height. In Figure 2 the same area was imaged in contact mode (A) and tapping mode (B). The steps are clearly visible in both modes, and each step is about 1nm high (see the line plot). This calibrated instrument was then used for all protein investigations. Imaging of Single Lysozyme Molecules. Figure 3 shows lysozyme molecules adsorbed to mica imaged in phosphate buffer in contact mode (A) and in tapping mode (B). The lysozyme molecules are randomly distributed over the surface. The lateral diameters of the single molecules are, due to tip b r ~ a d e n i n gabout , ~ ~ 20 nm. For contact mode imaging, it was necessary to use a pH of 4 to adsorb the molecule tightly enough to the surface to balance the lateral forces during scanning (Figure 3A). At pH 4, all free amino groups of the lysozyme molecule are positively charged and the negatively charged carboxyl residues are almost all protonated so that the electrostatic attraction between the molecule and the negatively charged mica surface is m a ~ i m i z e d . ~ lAt s ~this pH, the enzymaticactivityof lysozymeis very low.33J7The activity of hen egg white lysozymeas a function of pH has a roughly parabolic shape with a maximum between pH 6 and pH 8, falling off steeply toward pH 4 and pH 10. In contact mode it is therefore only possible to image a barely active lysozyme molecule. At higher pH, where the molecule would be active, imaging in contact mode resulted in pushing the molecules aside. In tapping mode in liquids (Figure 3B) lysozyme could be imaged easily at pH 6. No pushing of the molecules occurred. As shown in Figure 3 in contact mode (A, line plot) the protein lysozyme has the expected height of about 3 nm. The soft molecules of lysozyme appear about 6-8 nm in height in tapping (B, line plot). This is not due to the different pH between the sample in contact and tapping modes. Imaging lysozyme molecules in tapping mode in liquids at pH 4,5,6, and 7 revealed no significant height differences (unpublished data). Lysozyme molecules imaged at these pH values appeared about 6-8 nm in height. There are several possible explanations for this observation. Different interactions between tip and protein vs tip and mica substrate might be the reason for this apparently large height. The first is that the apparent height might be caused by the viscoelastic properties of the soft proteins.15 As the cantilever oscillates on top of a soft protein, the amplitude changes and leads to a different response of the error amplifier. To explain this, we would like to suggest a thought experiment. First we assume a physically homogenous sample with only topography changes, such as the scratched mica of Figure 1. Moving over a step (say from below to the top level), we find the distance of the cantilever base to the sample becomes smaller; thus, the amplitude of the resonating cantilever becomes smaller too. The sample will be retracted by the piezo until the original amplitude is restored, which will happen after a distance corresponding to the height of the step. Let us now assume a sample (30)Keller, D. Surf Sci. 1991,253,353. (31)Tilton, R.D.; Blomberg, E.; Cleasson, P. M.Langmuir 1993,9, 2102. (32)Van Wagenen, R. A,; Andrade, J. D. J. Colloid Interface Sci. 1980,76, 305. (33)Jolles, P.; Bernier, I.; Berthou, J.; Charlemagne, D.; Faure, A.; Hermann, J.; Jolles, J.; Perin, J.-P.; Saint-Blancard, J. In Lysozyme; Ossermann, E. F., Canfield, R. E., Beychok, S. Eds.; Academic Press: New York, 1974;p 31.

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Figure 4. G-actinadsorbed onto mica at pH 7 imaged in tapping mode in liquids. The molecules are randomly distributed over the surface and may be scanned with high forces (up to 500600 pN) without any visible damage.

with no topographic changes but with a spot where the interaction is strongly attractive between tip and sample. Here, over the attractive spot, the tip will be pulled down to the sample causing a decrease of the amplitude. The feedback will correct by retracting the sample slightly, which will appear in the image as if there is a topography feature present. On our proteidmica samples we have soft spots which show different interactions with the tip and which are also topography features. Therefore, the “height” signal can be a convolution of topography and tip-sample interaction causing an indentation of the sample in contact mode. A second kind of tip-sample interaction could be due to local differencesin charge due to electrostatic properties of the protein. This would again influence the error amplifier response. A third possibility would be that the soft protein molecules constantly move on a time scale of milliseconds (tapping frequency). A fourth possibility would be that there is a layer of weakly adsorbed ions and molecules surrounding the protein, thus increasing its apparent size for a gentle probe. More work will be necessary to determine which, if any, of these possibilitiesis the dominant effect. One possibility to test whether viscoelastic properties of the molecules are responsible for the apparent height is to fix the proteins with glutaraldehyde, for example. This was done with microtubulesand the apparent height decreasedto a lower value. Since the fixing with glutaraldehyde also affects the charge distribution of the protein (as it binds to free aminogroups), it is not yet clear which of the two phenomena leads to the increased height. Another possibility is to use a different tip chemistry, like electron beam deposited (ebd)34tips, to find out if a chemical interaction of the protein molecules with the tip might be the reason for the apparent height of the protein molecules. Imaging Monomeric Actin in Buffer Solution. Monomeric actin could be adsorbed onto freshly cleaved mica in a buffer solution with low ionic strength and at pH 7. The molecules are distributed randomly over the surface (Figure 4). Their measured lateral size is about 15 nm, larger than the diameter of G-actin of about 5.5 (34)Keller, D.; Deputy, D.; Alduino, A.; Luo, K. Ultramicroscopy 1992,42-44,1481-1489.

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Figure 5. Actin filaments adsorbed onto mica imaged an AFM tapping mode in liquids. Part A shows a large scan area where the filaments are randomly distributed. In (B) a closeup view of one filament is shown. The helix pitch of the two-strand helix is readily seen. Thesaveragehelix turn is 37 nm (see also inset), which is in agreement with TEM and X-ray data.

nm determinedby X-ray crystallography.20 This is again due to tip broadening. Although the isoelectric pH of G-actin is about 6, it was possible to adsorb the protein to negatively charged mica indicating that the interaction between the G-actin molecules and the mica surface is not purely electrostatic. The molecules again appear about twice as high (10- 14 nm) as the expected height of about 6 nm from crystallographic data. Imaging Actin Filaments in Physiological Buffer Solution. Figure 5 shows actin filaments that were adsorbed onto mica. A solution of 0.5 mg/mL of monomeric actin was polymerized in F-buffer at 4 "C for 12 h. The substrate mica was freshly cleaved, and 50 ,uL of the filament solution was incubated for 30 s on the substrate. After flushing the unbound molecules vigorously away with the same buffer at pH 6, the sample was imaged at room temperature using tapping mode in liquids. In Figure 5A a typical large scan area is shown. The filaments are randomly oriented on the surface. They are 50 nm wide, which is due to the finite size of the tip. Their height is about 20 nm and, therefore again, higher than the expected value of 10 nm. Stable imaging was possible at forces below about 200 pN. At this low force the filaments could be imaged for more than 10 frames without any visible damage. Features within the filament are nicely visible (Figure 5B). The helical turn in the filament spanning 13 monomers is resolved. The helix appears as a two-strand right-handed helix. The average length for one turn is 37 nm (see inset and line plot, Figure 5B). This is in good agreement with data from electron m i ~ r o s c o p y 3and ~~~~ from X-ray fiber diagrams from oriented F-actin gels.37 Imaging with higher forces (200pN) removed the upper actin monomers in the filament which are not attached to mica. The filament only remained intact in a few areas (thick arrows). Those monomers which are boynd to the mica surface are still in place (thin arrows).

action is stronger than the monomer-monomer interaction at the given buffer. Imaging Microtubules in Buffer Solution. Microtubules at pH 7 are negatively charged hollow cylinders. Their isoelectrical pH is about 5.5. The number of protofilaments in the circumference is in vivo almost always 13 but varies with buffer conditions in vitro. The outer diameter of a filament is about 30 nm. Some microtubule cylinders are rigid structures, they appear as straight filaments that show hardly any bending on the scale of a few micrometer^;^^ their length can be several (38)Venier, P.;Maggs, A. C.; Carlier, M.-F.; Pantaloni, D. J. Biol. Chem. 1994,269,13353.

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mica in physiological buffer with slightly lower pH (like pH 6) with applied loading forces of less than 200 pN. The helix turn can be clearly resolved and is 37 nm in the adsorbed form. With loading forces greater than 200 pN the upper actin monomers in the filament can be removed from the filament, showing the adsorbed monomers still in their filamentous arrangement. Microtubules do not adhere to negatively charged surfaces but can be imaged on a positively charged silane coated on glass. Imaging actin filaments revealed the resolution of the helix pitch, and under certain circumstances, of single monomers along the length of a filament. Microtubules appear smooth in the height image. In tapping mode in liquids, solid surfaces can be used to calibrate the height signal whereas proteins show an apparent height which might be due to viscoelastic properties or to electrostatic properties of the proteins. For all the proteins, lysozyme, actin, and microtubules the height is larger than expected. Treating microtubules with glutaraldehyde leads to a height slightly lower than their expected height. More work will be necessary to determine the effect of the apparent height in imaging proteins in tapping mode in liquids. Figure 7. Microtubules adsorbed on glass slides coated with DETA imaged an AFM tapping mode in liquids. They bind strongly to the positively charged surface. The hollow cylinders are stiff rods and therefore adsorb straight onto the substrate without bending.

millimeters. We could not image filaments directly adsorbed to the negatively charged surfaces of mica or glass. We needed to use a positively charged surface: trimethoxysilylpropyldiethylenetriamine (DETA)(Jeffrey Calvert, personal communication). On the DETA-coated glass surface it was possible to image microtubules with high resolution. The positively charged amino groups can interact with the acidic residues on the microtubules shown in Figure 7. Clearly visible are the straight strands which do not bend on this length scale. They can be imaged, even with forces above 400 pN, without any visible damage or change. Microtubules which are polymerized with taxol in the buffer solution and then adsorbed to the DETA surface appear also, as with the other investigated proteins, about twice as high as the expected 30 nm. After glutaraldehyde treatment their height appears to be about 20 nm and thus smaller than the expected 30 nm (data not shown). Since glutaraldehyde might affect various types of tip-sample interactions the nature of the apparent height cannot be explained yet.

Conclusions By use of tapping mode in liquids, globular proteins can be imaged stably in buffer solution, without any visible damage. The globular proteins lysozyme and G-actin adsorb strongly enough to freshly cleaved mica to be imaged at physiologicalpH without any further treatment. The filamentous proteins were more difficult to image. Actin in its filamentous form can be imaged adsorbed to

Abbreviations atomic force microscopy adenosine triphosphate trimethoxysilylpropyldiethylenetriamine dithiothreitol ethylene glycol bis(/3-aminoethylether)-N,N,”,”tetraacetic acid F-actin filamentous polymeric actin G-actin globular monomeric actin GTP guanidine triphosphate isoelectric point PI PIPES piperazine-N,”-bis( 2-ethanesulfonic acid) TEM transmission electron microscopy

AFM ATP DETA DTT EGTA

Acknowledgment. We thank Neil Thomson for carefully reading the manuscript. We thank Dan Morse, Helen Hansma, Jan Hoh, Klaus Schulten, Erich Sackmann, and Herrman Gaub for helpful discussions and Ed deLong for the use of his table-top ultracentrifuge. The work was supported by the Materials Research Program of the National Science Foundation under Grant No. NSF DMR 9123048 (P.H.), the Material Research Division under Grant No. NSF DMR 221781 (M.R.), the Office of Naval Research under Grant No. ONR NO00149251260 (J.C.), NIH Grant AR38910 (P.J., and R.G.), The Whitakes Foundation (M.W.A., C.F.S.), Exxon Education Foundation (C.F.S.), NIH Molecular Biophysics Training Grant (M.W.A.), and the Deutsche Forschungsgemeinschaft (M.F.). We thank Digital Instruments for supporting us with instrumentation. LA9502397