Center of Competence in Research (NCCR) TransCure, and the. European Science Foundation (Grant 09-EuroSYNBIO-FP-012. NANOCELL to D.F. and D.J.M.) ...
Chapter 24 High-Resolution Imaging of 2D Outer Membrane Protein F Crystals by Atomic Force Microscopy Dimitrios Fotiadis and Daniel J. Müller Abstract In this chapter the methodological bases are provided to achieve subnanometer resolution on two-dimensional (2D) membrane protein crystals by atomic force microscopy (AFM). This is outlined in detail with the example of AFM studies of the outer membrane protein F (OmpF) from the bacterium Escherichia coli (E. coli). We describe in detail the high-resolution imaging of 2D OmpF crystals in aqueous solution and under near-physiological conditions. The topographs of OmpF, and stylus effects and artifacts encountered when imaging by AFM are discussed. Key words: Atomic force microscopy, Biological membranes, High resolution, Outer membrane protein F, Stylus artifacts, Topography
1. Introduction In more than 20 years of continuous technological and methodological development the AFM (1, 2) has become a powerful multifunctional nanoscopic tool to image and to sense a variety of cell and molecular biological systems (3–8). The unique features of imaging biological structures in their physiologically relevant buffer solution, temperature and pressure, the high lateral and vertical resolution, and the high signal-to-noise ratio of the AFM topographs make this microscope outstanding. Today, AFM is routinely applied to directly contour the topography of complex biological systems across dimensions ranging from tissues to cells and towards the substructure of single proteins. Whereas in the imaging mode the AFM stylus is used to contour the topography of the native biological sample, in the sensing mode the AFM stylus
Ingeborg Schmidt-Krey and Yifan Cheng (eds.), Electron Crystallography of Soluble and Membrane Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 955, DOI 10.1007/978-1-62703-176-9_24, © Springer Science+Business Media New York 2013
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is applied to probe chemical, biological, or physical interactions at molecular resolution. As a contouring and probing tool the AFM is particularly suited to image biological surfaces such as those of living cells, cell membranes, and reconstituted membrane proteins. When applied to biological membranes, high-resolution AFM has allowed the observation of the assembly, oligomeric state, diffusion, and function of native single membrane proteins. Complementary, the sensing mode AFM enables the user to spatially correlate and quantify biochemical and physical properties of membrane proteins, such as electrostatic potential, stability, flexibility, elasticity, conformational entropy, folding and unfolding pathways, and ligand- and inhibitor binding (for recent reviews see refs. 6, 9–12). From this variety of possible applications of AFM to characterize membrane proteins (4, 13), this chapter focuses on the application of contact mode AFM imaging to acquire high-resolution structural information of native membrane proteins in buffer solution. In contact mode AFM, a nanometer-sized sharp stylus mounted on a 50–400 μm long flexible cantilever is raster scanned, thereby contouring the sample topography at constant force. The topography is reconstructed from the up-and-down displacements of the cantilever recorded as the stylus is scanned. To minimize possible damage of the biological specimen by the AFM stylus, soft cantilevers with spring constants around 0.1 N/m are preferred and scanning must be performed at minimal force applied to the stylus (~50–100 pN). Lateral resolutions better than 0.5 nm and vertical resolutions approaching 0.1 nm have been achieved on biological membranes in solution (7, 10, 11). Up to now this AFM imaging mode has provided the highest resolution with biological samples. Alternative AFM modes to record topographs are the oscillation mode (14–18) and the frequency modulation mode (19–21). The oscillation mode is frequently used to contour the surface topography of weakly attached biomolecules, i.e., single proteins, fibrils, and chromosomes, by vertically oscillating the AFM stylus while scanning the sample. Because the oscillating AFM stylus touches the sample surface only at the end of its downward movement, the frictional forces are reduced, avoiding the lateral deformation and displacement of the sample. In contrast to contact and oscillation mode, in the frequency modulation mode the AFM stylus hardly touches the sample surface as it senses interactions that are not based on the formation of a physical contact with the sample. Although having already provided promising results on membrane proteins (22), this AFM mode is still in its infancy for biological AFM imaging approaching a lateral resolution of 96% (v/v)). 6. Loctite 406 superglue from KVT König, Dietikon, Switzerland. 7. Araldit Rapid: Two-component epoxy glue from Tesa AG, Bergdietikon, Switzerland. 8. Scotch tape.
2.2. OmpF Porin and Buffers
1. OmpF from E. coli was purified, reconstituted into lipids, and crystallized in 2D as described (30). Stock solution of OmpF 2D crystals for AFM experiments: 1 mg/ml in 20 mM HEPES– NaOH (pH 7), 100 mM NaCl, 2 mM MgCl2. Store at 4°C. 2. Adsorption and imaging buffer: 20 mM Tris–HCl (pH 7.8), 300 mM KCl (31). Prepare buffers for AFM freshly. 3. Prepare all buffers using nanopure water (~18 MOhm/cm) that has been filtered through nanometer sized pores.
2.3. AFM and Accessories
1. A commercial AFM equipped with a >100 μm piezoelectric scanner and a liquid cell (e.g., NanoWizard, JPK Instruments, Berlin, Germany or Multimode, Veeco Metrology Group, Santa Barbara, CA, USA). 2. Oxide-sharpened Si3N4 microcantilevers of 100 μm length and a nominal spring constant of k = 0.08 N/m (Olympus Optical Co., LTD, Tokyo, Japan).
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Imaging 2D crystals of OmpF by AFM
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3. Method 3.1. Preparation of Mica Supports for Sample Immobilization
1. Punch mica disks of 6 mm and Teflon disks of 14 mm diameter using the “punch and die” set and a hammer. 2. Clean the Teflon and steel disks with ethanol and paper wipes. 3. Glue a Teflon disk on a steel disk with Loctite 406. 4. Glue a mica disk on the Teflon surface of the Teflon-steel disk using the two-component epoxy glue Araldit. 5. Let the glued supports strengthen for at least 1 day.
3.2. Adsorption of 2D OmpF Crystals to Mica
1. Dilute and mix 2 μl of 2D OmpF crystal stock solution with 40 μl adsorption buffer in an Eppendorf tube. Typical final protein concentrations are between 20 and 60 μg/ml. 2. Cleave mica with Scotch tape. 3. Pipet 20–30 μl of the diluted OmpF 2D crystals onto the freshly cleaved mica support. 4. Adsorb OmpF for 15–30 min. 5. Rinse the mica to remove weakly attached 2D crystals by aspirating approximately 2/3 of the fluid volume from the mica surface and re-adding the same amount of imaging buffer. Repeat this washing procedure at least three times. 6. Transfer and mount the mica support to the AFM. 7. Mount the cantilever to the fluid cell (preferably without o-ring seal to minimize drift) and mount the fluid cell to the AFM. 8. Fill the space between the mica surface and the fluid cell with imaging buffer to avoid dehydration of the sample.
3.3. Operation of the AFM
1. After thermal relaxation of the AFM, initial engagement of the AFM stylus is performed. Set the scan size to 0 to avoid that the approaching AFM stylus starts scanning and becomes contaminated by the biological specimen. Prior to scanning the sample surface, the operating point of the instrument is set to forces below 0.5 nN. For overview imaging at low magnification (frame size >1 μm) the forces are kept below 0.5 nN and for high-resolution imaging below 0.1 nN (frame size 1 μm), whereas height signals are acquired in both, trace and retrace scanning direction when approaching higher magnification (frame size
