Langmuir 2000, 16, 5993-5997
5993
Single Molecule Height Measurements on a Membrane Protein in Nanometer-Scale Phospholipid Bilayer Disks Timothy H. Bayburt,†,‡ Joseph W. Carlson,†,§ and Stephen G. Sligar*,†,‡,§ Beckman Institute for Advanced Science and Technology, Department of Biochemistry, and Center for Biophysics, University of Illinois, Urbana, Illinois 61801 Received November 4, 1999. In Final Form: March 24, 2000 Atomic force microscopy can be used to determine the vertical dimension of biological molecules under native conditions with high resolution. Deformation of soft proteins by the scanning force, however, introduces error in the magnitude of the measurement. In this work, the force-dependent height of NADPH-cytochrome P450 reductase, an integral membrane protein, was measured by atomic force microscopy and analyzed to account for the contribution of deformation to the observed height of the molecule above a model membrane surface. Imaging of single reductase molecules was accomplished by reconstitution into 10 nm diameter phospholipid bilayer particles, which provides a way of adsorbing the protein-phospholipid complex on a surface in the proper orientation. The results show that the height of the reductase is drastically underestimated in contact imaging mode. An analysis of force curves taken on single reductase molecules provides a height that better matches the known dimensions of the protein. This technique should be generally useful for determining the vertical dimension of biological samples that are severely deformed by contact imaging forces.
Atomic force microscopy (AFM) uses the interaction of a tip, with a radius of curvature of 5-40 nm, attached to a flexible cantilever to obtain a topographical image of a sample surface. An inherent difficulty in AFM is the force required for imaging. Soft biological samples deform under the force of the tip, leading to error in physical measurements of the size of the molecules under study. This effect is even more predominant when single molecules are being studied in the absence of stabilization, such as without crystallization or fixation. Imaging at cryogenic temperatures1,2 has been used to overcome some of these problems but requires sophisticated instrumentation. Measurement of cantilever deflection while the sample remains stationary in a lateral plane is often used to obtain force-distance measurements. The cantilever deflection, combined with its known spring constant, allows interpretation of images with respect to the applied force. Most reported AFM images, however, are usually obtained at a single force. Collection of consecutive images at different forces is possible but difficult due to the drift in applied force during data acquisition.3,4 However, complete force curves may be obtained over a large imaging area, a technique called force mapping.5 In contrast to images obtained at a single low force, images obtained by measuring force curves over the sample area contain information about the force-dependent topography of the sample from a finite to an absolute force of zero. These approaches have been used to determine the viscoelastic * To whom correspondence should be addressed: Beckman Institute, University of Illinois, 405 N. Mathews, Urbana, IL 61801. Tel: 217-244-7395. Fax: 217-244-7100. E-mail:
[email protected]. † Beckman Institute for Advanced Science and Technology. ‡ Department of Biochemistry. § Center for Biophysics. (1) Zhang, Y.; Sheng, S.; Shao, Z. Biophys. J. 1996, 71, 2168-2176. (2) Han, W.; Mou, J.; Sheng, J.; Yang, J.; Shao, Z. Biochemistry 1995, 34, 8215-8220. (3) Frizsche, W.; Henderson, E. Ultramicroscopy 1997, 69, 191-200. (4) Radmacher, M.; Fritz, M.; Hansma, P. K. Biophys. J. 1995, 69, 264-270. (5) Radmacher, M.; Fritz, M.; Cleveland, J. P.; Walters, D. A.; Hansma, P. K. Langmuir 1994, 10, 3809-3814.
properties of human platelets,6 cells,7-9 lysozyme,5 and polymer films.4 We have previously reported the use of 10 nm scale phospholipid bilayer structures for the imaging of singlemembrane proteins.10 The structure consists of a discoidal phospholipid bilayer domain stabilized at the edges by a protein shell of apoA-I, which forms amphipathic R-helices that associate with the hydrophobic edges of the phospholipid bilayer disk. The conclusion that the objects seen in AFM images were reductase molecules associated with the discoidal structures through their membrane anchors and in the proper orientation on the sample surface was based on the following evidence:10 (1) The objects were not observed if rHDL was formed in the absence of P450 reductase. (2) P450 reductase activity was present on the mica surface in approximately the amount necessary to account for the observed number of objects. (3) P450 reductase is associated with 10 nm diameter rHDL particles in solution and can be removed using light trypsin treatment which is known to cleave between its membrane anchor and a large hydrophilic catalytic domain.11 Trypsin treatment of the rHDL/P450R surface removes the objects seen in AFM images while rHDL particles are resistant to trypsin treatment. (4) The underlying surface is the bilayer domain of rHDL because the domains can be induced to fuse and form a smooth continuous surface having the thickness of a single phospholipid bilayer. The advantages of nanometer-diameter discoidal high-density lipoprotein (rHDL) phospholipid domains over other surface-bound systems include the self-formation of a surface of oriented assemblies from solution, the ability to pattern nanometer-sized domains of varying size and (6) Radmacher, M.; Fritz, M.; Kacher, C. M.; Cleveland, J. P.; Hansma, P. K. Biophys. J. 1996, 70, 556-567. (7) Ricci, D.; Grattarola, M. J. Microsc. 1994, 176, Pt 3, 254-261. (8) Hoh, J. H.; Schoenengberger, C.-A. J. Cell Sci. 1994, 107, 11051114. (9) Haydon, P. G.; Lartius, R.; Parpura, V.; Marchese-Ragona, S. P. J. Microsc. 1996, 182, 114-120. (10) Bayburt, T. H.; Carlson, J. W.; Sligar, S. G. J. Struct. Biol. 1998, 123, 37-44. (11) Phillips, A. H.; Langdon, R. G. J. Biol. Chem. 1962, 237, 26522660.
10.1021/la991449c CCC: $19.00 © 2000 American Chemical Society Published on Web 06/17/2000
5994
Langmuir, Vol. 16, No. 14, 2000
shape, and the presence of apolipoprotein A-I (apoA-I) at the bilayer edges which might be engineered to provide additional desired functionalities.10,12 The underlying discoidal structures containing a membrane protein are easily recognizable and provide a point of reference for judging the quality of the sample and images. One fundamental goal in structural studies of membrane proteins is to determine their position with respect to the cell membrane. Knowledge of the position of membrane-associated proteins with respect to the membrane bilayer is important for understanding their structure, function, and interaction with target molecules. The present work describes a direct method based on the use of force curves to measure the distance single proteins protrude from a membrane surface with a scanning force microscope. Our studies use the enzyme NADPH-cytochrome P450 reductase which is the flavoprotein redox donor to the P450 monoxygenases in the endoplasmic reticulum. A crystallographic model of the truncated catalytic domain lacking an N-terminal sequence containing the membrane anchor is available.13 Using the force-dependent topographic information, we determined the approximate position of the native membrane-bound enzyme normal to the bilayer. In contrast to measurements taken at low constant force, the value obtained by analysis of cantilever deflection as a function of tip-sample separation is consistent with the known structure of the reductase. Experimental Procedures Materials. Human apoA-I, purified as described,14 was the kind gift of Dr. Ana Jonas (University of Illinois, ChampaignUrbana). ApoA-I migrated as a single band on a Coomassiestained SDS-PAGE gel. NADPH-cytochrome P450 reductase was overexpressed in E. coli and purified according to a previously published procedure.10,15 Cytochrome P450 reductase concentration was determined from absorbance at 456 nm using an extinction coefficient of 21 400 M-1 cm-1.16 Specific activities were in the range of reported values (45-52 µmol/(min/mg)). The purified reductase was observed to run as a single band on Coomassie-stained SDS-PAGE gels. Cytochrome P450 reductase was reconstituted into rHDL by the detergent dialysis method as described previously.10 Dipalmitoyl phosphatidylcholine was obtained from Avanti Polar Lipids. Muscovite mica was obtained from S and J Trading Inc. (Glen Oaks, NY). Silicon nitride AFM tips were from Digital Instruments (Santa Barbara, CA). Water used for reconstitution of rHDL and imaging was purified with a Milli-Q system (Millipore, Bedford, MA). Atomic Force Microscopy. AFM images were obtained with a Nanoscope IIIa (Digital Instruments, Santa Barbara, CA) equipped with a fluid cell using the “A” scanner. The calibration of piezo movement in the z-direction was initially performed using 180 nm deep pits in a silicon standard (Digital Instruments) and fine-tuned by measuring the 1 nm periodicity of mica cleavage planes. To form the surface of rHDL/cytochrome P450 reductase, mica was glued to 10 mm steel disks and cleaved with cellophane tape and 2-10 µL of rHDL/cytochrome P450 reductase was applied followed by 10-20 µL of imaging buffer (10 mM Tris‚ HCl pH 8.0, 0.15 M NaCl, 10 mM MgCl2). Addition of rHDL/ cytochrome P450 reductase after buffer gives identical results. The use of a PAP pen (Ted Pella Inc., Redding, CA) to circumscribe an area of mica with a hydrophobic border has been found useful to prevent flow of solution off the mica. After 30 min, the sample (12) Carlson, J. W.; Jonas, A.; Sligar, S. G. Biophys. J. 1997, 73, 1184-1189. (13) Wang, M.; Roberts, D. L.; Paschke, R.; Shea, T. M.; Masters, B. S. S.; Kim, J. P. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8411-8416. (14) Jonas, A.; Ke´zdy, K. E.; Wald, J. H. J. Biol. Chem. 1989, 264, 4818-4824. (15) Shen, A. L.; Porter, T. D.; Wilson, T. E.; Kasper, C. B. J. Biol. Chem. 1989, 264, 7584-7589. (16) French, J. S.; Coon, M. J. Arch. Biochem. Biophys. 1979, 195, 565-577.
Bayburt et al. was mounted in the fluid cell and several milliliters of imaging buffer was passed through the cell to remove any unadsorbed material. Contact imaging was done under imaging buffer using the thin-legged 200 µm cantilever having a nominal spring constant of 0.06 N/m. Force Curves and Data Analysis. Force curves were obtained using the force-volume feature of the Nanoscope IIIa data acquisition software. Calibration of cantilever deflection was performed by taking force curves over a region of the rHDL surface. Cantilever deflection calibration is best obtained by taking a force curve on a hard surface. In our case, calibration of the deflection offset voltage was performed over the rHDL surface which shows nonlinearity at lower deflection values. This approximation is rationalized by the fact that the calibration used the linear portion of the contact region at the higher deflections typically achieved and that in this linear region the slope on rHDL is similar to that of force curves taken on a mica surface. For example, a slope of -1.0 ( 0.05 was found on rHDL, compared to a slope on mica of -1.0 ( 0.01 obtained by a leastsquares fit between deflection values of 10 and 15 nm after calibration of the deflection voltage on the rHDL surface. Force curves were measured using the “relative trigger” mode in which the maximum cantilever deflection is constant. The trigger value was set to 15 or 20 nm. Sixty-four samplings were taken for each force curve, and each force-volume image was comprised of an array of 64 × 64 force curves over a 500 × 500 nm area. Data were exported to a spreadsheet for analysis. Only force curves in the approach direction were analyzed. Force curves were filtered using the Savitzky-Golay quadratic smoothing algorithm17 to reduce noise. The slope and y-intercept for each force curve were calculated from the 10 largest deflection values (between 10 and 15 nm deflection or higher). An averaged theoretical force curve for a hard surface was obtained by averaging the intercept values and the slopes. The line defined by these parameters represents the contact portion of a force curve on a hard surface, which should have a negative unity slope and an intercept value defined by the trigger threshold value. The slope and intercept values did not differ markedly (
