Proc. Nati. Acad. Sci. USA
Vol. 89, pp. %32-9636, October 1992 Biophysics
Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor (synaptic membrane/surface dimensions/central cavity)
A. BERTAZZON, B. M. CONTI-TRONCONI, AND M. A. RAFTERY* Department of Biochemistry, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108
Communicated by David S. Eisenberg, May 15, 1992 (received for review July 19, 1991)
The synaptic surface of the acetylcholine reABSTRACT ceptor in membranes from Torpedo calzfornica electric organ has been imaged by anig tunneling microscopy. The molecule appears pentameric, with one major and four minor protrusions rising above the surface, and these protrusions encompass a large central cavity. The outer diameter of the molecule is 69 ± 10 A, while the diameter of the cavity, measured at the widest complete contour line delmiting the opening, is 26 ± 7 A. The images and dimensions obtained are consistent with the structure determined from hybrid density maps obtained by x-ray diffraction and electron microscopy. Thus, scanning tunneling microscopy can be used to obtain overall dimensions and low-resolution structural features of the surface of a membrane-embedded protein.
Given the difficulties encountered in preparing crystals from membrane proteins suitable for x-ray or electron diffraction analysis, it is worthwhile to explore other methods for obtaining structural information on these rather intractable systems. The introduction of scanning tunneling microscopy (STM) for imaging of biological systems (1) can yield structural data at reasonably good resolution. This method is capable of atomic resolution for smooth and highly conductive surfaces of metallic compounds but not, at least now, for biological samples, which are considered poor conductors. Many such studies are currently under way (2). We wished to test the application of STM to biological membranes by using a system for which some characteristic features of surface structure are known. The system should ideally be composed of a large protein embedded in a biological membrane that is homogeneous with respect to protein composition. Such criteria are met by postsynaptic membrane preparations from Torpedo species such as Torpedo californica because they can be readily prepared (3, 4) and have been shown to contain the nicotinic acetylcholine receptor (AChR) with minimal contamination by other proteins (5). This AChR is a large protein complex (270 kDa) composed of five subunits (6, 7) with the stoichiometry a213y6 (6, 8). A three-dimensional model of Torpedo AChR, based on density maps obtained from low-dose electron microscopy images and x-ray diffraction at 12.5-A resolution, has been described (9). The AChR protrudes 55 A from the outer surface of the lipid bilayer and the average diameter of this extracellular part (50-60%o of the total mass) of the AChR molecule is 65-80 A (10). The view from above the membrane of a sample negatively stained for electron microscopy shows a rosette with a central cavity of diameter 25-30 A (11, 12). The map further shows that five electron-dense regions make up the rosette, possibly corresponding to the five constituent subunits (9), with a characteristic protrusion of density rising above the other regions (10). This preparation of a large
FIG. 1. STM image of AChR-rich membrane fragments from electric organ of T. californica. Two receptor molecules (arrows) are visible. The original image was taken by scanning a field of 230 x 230 nm at a probe scan rate of 1400 nm/sec, sampling time of 18 jusec, tunneling current of 0.56 nA, bias voltage of 32 mV, and integral and proportional gains of 2.0 and 3.0, respectively. The enlargement has been filtered using the x, y plane fit and the flatten functions. Color scale is in nanometers. Backgrounds ofall preparations revealed only the atomic structure of the support.
integral membrane protein whose dimensions and general structural features are known represents a suitable system for application of STM to study the surface structure of a membrane protein. We report here images obtained from different preparations of T. californica AChR in enriched membranes, an analysis of the outer surface dimensions, and a comparison ofthe results with those obtained from electron microscopic and x-ray analysis studies.
MATERIALS AND METHODS Membrane Purification. AChR-rich membranes were prepared from T. californica electric organ (13) and treated at pH 11 (14, 15) to remove extrinsic membrane proteins. The protein subunit composition was assessed by SDS/PAGE (16). Binding activity was determined by use, of 125I-labeled a-bungarotoxin and a DEAE disk assay (17). Octyl f3-Dglucopyranoside (1.0%, wt/vol) was used to solubilize the AChR, followed by centrifugation at 100,000 x g for 1 hr to
eliminate undissolved debris. AChR preparations were stored at 40C and used within a few days.
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Abbreviations: STM, scanning tunneling microscopy, AChR, acetylcholine receptor; HOPG, highly oriented pyrolytic graphite. *To whom reprint requests should be addressed. %32
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Proc. Natl. Acad. Sci. USA 89 (1992)
FIG. 2. Three-dimensional view of a single AChR molecule. (Upper) A filtered three-dimensional view of a single AChR molecule. (Lower) Top view of an unfiltered (Left) and a filtered (Right) image. Flattening and plane fitting reduce the sharpness of the image and result in a loss of detail, but they provide consistent images for determination of the average dimensions reported in Table 1. A pentagonal shape, with a typical protrusion and a central cavity, can be recognized. The lowest part of the surface is consistently localized on both sides of the major peak (bottom of the image). The original image was obtained at 50-mV bias voltage, 1.9-nA tunneling current, and scan rate of 3.2 cycles per second on a field 250 x 250 nm (1600 nm/sec). Color scale is in nanometers. In membrane preparations poor in AChR content no images were observed except for uninterpretable structures.
Instrumentation. Scans were performed with a Nanoscope (Digital Instruments, Santa Barbara, CA) equipped with a size D head, 0.8-pim scanning field. Probes were cut from 0.25-mm-diameter Pt/Ir (80:20) wire with sharp scissors or purchased from Digital Instruments (Nanotips). Highly oriented pyrolytic graphite (HOPG) was purchased from Union Carbide (Cleveland) (10 x 10 x 0.2 cm). Sample Deposition. AChR-enriched membranes were suspended in 10 mM Tris HCl at pH 7.4 or pH 9.5. Membranes were stored at pH 7.4 and the pH was adjusted to 9.5 not more than 30 min before deposition onto HOPG. All samples were diluted to a protein concentration of 0.1-0.3 mg/ml in 10 mM Tris HCl. Membrane deposition was achieved by loading 5 Al of the solutions onto a clean, newly cleaved surface of HOPG and spread over an area of about 0.2 cm2. Snning Parameters. All scans were performed in air at room temperature. The quality of the tip was checked by its ability to properly image the graphite background. Typical starting bias voltages were 30-50 mV, with the positive direction toward the tip (current flow from sample to tip). Large fields (1-5 ,m) were scanned at a rate of 2-5 cycles per second using a bias voltage between 20 and 100 mV, with tunneling currents ranging from 0.4 to 2 nA, integral gain set from 2 to 6 and a proportional gain ranging between 3 and 9 to maintain a constant ratio of 1.5 with the integral gain; the two-dimensional gain was 0.05. Data analysis and image II
filtering were performed with software provided by Digital Instruments. High-frequency noise was removed by using the lowpass function, image curvature was removed by using the plane fit function, which removes, in both x and y directions, a least-square fit first-, second-, or third-order polynomial from the data. Where the sample is imaged there is no visible background. In areas with no sample, the HOPG can be clearly imaged at 10-nm field size; resolutions at 0.8-nm field showing the typical graphite hexagon are obtained only in areas of the graphite surface where the sample was not deposited. Table 1. Average dimensions of the AChR (A) Ref. 7 This work* Ref. 6 65 72 69 ± 10 (12) Outer diameter 25 30 26 ± 7 (15) Inner diametert 9 ± 4 (13) Majorpeak* *Dimensions were obtained from filtered images and are expressed in angstroms (mean ± SD). In parentheses is the number of molecules used. The images are stored on discs and can be furnished to readers who are interested. tMeasured from contour maps (as in Fig. 3) and corresponding to the largest diameter measured on the first complete contour line delimiting the central cavity. *Height of the major peak measured from the first contour line.
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FIG. 4. CD spectra of resuspended, solubilized AChR. Membrane-bound AChR was deposited onto graphite and allowed to dry. After 30 min (curve 2) and 3 hr (curve 3) the AChR was solubilized in 10 mM Tris-HCI containing 1% octyl 3-D-glucopyranoside. Curve 1 was obtained from a membrane preparation in 10 mM Tris-HCl solubilized by addition of 1% octyl 3-D-glucopyranoside. Values obtained from analysis of the spectra are shown in Table 2.
FIG. 3. Subunit localization on unfiltered images of single AChR molecules. The general pentameric geometry observed in filtered images is generated by five separate peaks on the surface. (Upper) Enlargement from a 193 x 193-nm field, obtained with a bias voltage of 18 mV, a tunneling current of 1.9 nA, a scanning rate of 1.3 cycles per second (650 nm/sec), and sampling of 18 lsec. The contour lines are spaced 0.3 nm in the vertical dimension. Color scale is in nanometers. (Lower) Image obtained from a 230 x 230-nm field with instrumental settings as above and a scanning rate of 2.7 nm/sec. Five areas can be distinguished.
Receptor Stability. The conditions used for STM sample deposition were also employed for binding and structural experiments. Receptor suspensions (150 IlI) in 10 mM Tris-HCl were allowed to dry on HOPG surfaces. Dried samples were solubilized in 150 ,u of 10 mM Tris-HCl containing 1% octyl ,B-D-glucopyranoside. Control experiments were performed with the same preparation simply solubilized by adding octyl f-D-glucopyranoside to a final concentration of 1%, without deposition or drying. Circular Dichroism (CD). Spectra were taken with a Jasco J 41 C spectropolarimeter (Jasco, Easton, MD) interfaced through an Adalab A/D converter (Adalab-PC Interactive Microware, State College, PA) to an IBM-XT compatible computer. The instrument was calibrated with (+)-10camphorsulfonic acid. Water-jacketed thermostatted quartz cuvettes with 0.1- and 1-mm path lengths were obtained from Hellma (Forest Hills, NY). Optical activity, expressed as mean residue elipticity (6) in degrees-cm2 dmol-1, was analyzed by means of a constrained nonlinear least-square fit (18).
RESULTS When AChR-enriched membranes were deposited on the HOPG substrate at pH 7.4 they had a tendency to aggregate in layers 100-150 nm thick and were readily swept away by the scanning tip, with formation of new aggregation patterns. However, when the sample was deposited at pH 9.5 such aggregates were not observed and the membranes consistently formed layers 30-50 nm thick. An enlargement of a 15 x 15-nm scanning field is shown in Fig. 1. The image was taken at a scanning rate of 1400 nm/sec (3.2 cycles per second; field, 230 x 230 nm), at a bias voltage of 32 mV, a tunneling current of 0.56 nA, integral and proportional gains 2.0 and 3.0, respectively, and the enlargement was filtered using the plane fit (automatic x and y) and flatten functions. Two rosette-like structures, protruding 30-40 A from the membrane surface, were observed (center and upper right in Fig. 1) with an irregular surface and typical asymmetric protrusions. A three-dimensional view of a single receptor molecule is shown in Fig. 2 Upper. These images can be filtered as described above, and this results in a loss of detail but allows a standardized determination of average dimensions (Fig. 2 Lower). The dimensions obtained on several different samples are presented in Table 1. The average outer diameter is 69 10 A, while the central cavity, taken on contour maps offiltered images at the largest delimiting line, is 26 7 A, and the major protrusion, calculated from the lowest point of the receptor surface (typically on the side of the major peak), is ±
Table 2. Secondary structure analysis a-Helix p-Sheet (-Turn Random coil Sample This work Control membranes 38.6 32.1 1.2 28.1 33.2 32.6 30-min drying 33.4 0.8 3-hr drying 32.6 38.1 0.6 28.7 Literature 34 37 29 0 a* bt 35 24 10 31 22 41 8 29 ct The analysis was carried out with the algorithm of Chang et al. (18). *From Moore et al. (19) tFrom Wu et al. (20) tFrom Mielke and Wallace (21), referred to the constrained leastsquare fitting according to Chang et al. (18)
Biophysics: Bertazzon et al. Table 3. Binding of 1251-labeled a-bungarotoxin to solubilized AChR n Drying time, min Binding, % control 4 15 82 ± 7 4 30 87 ± 8 4 60 79±11 4 180 92 ± 4 One hundred fifty microliters of a receptor suspension, containing protein at 0.3 mg/ml, was allowed to dry on a newly cleaved HOPG surface. At defined time intervals the dried sample was solubilized in the same volume of 10 mM Tris-HCI, pH 7.4/1% octyl 3-Dglucopyranoside. Binding assay was performed as described (17). Control binding was obtained with solubilized membranes and was 2-4 nmol of 125I-a-bungarotoxin binding sites per milligram of protein.
9 ± 4 A. These data, presented in Table 1, compare nicely with other published values for the outer surface dimensions of the AChR molecule. Fig. 3 Upper shows a contour map of the unfiltered image of a single molecule, with the distance between lines being 3 A (vertical dimension). The total height above the background is 50 A, which is close to the dimensions reported earlier for the extracellular moiety of the molecule (9-12) protruding from the plane of the membrane. Five peaks can be observed, although two on the left side are not well resolved. The major peak protrudes about 15 A from the largest contour line delimiting the central pit, while the peak on the opposite side of the central opening (at top bar in Fig. 3) protrudes -9 A. Of the two peaks poorly resolved on the left side, the one on the lower left of the image protrudes 3 A and the one on the upper left 9 A above this same plane. On the right side a peak protruding -6 A is observed. This contour image closely matches those previously reported from hybrid maps (9). The average width of the walls of the pseudosymmetric rosette surrounding the central pit is 25 A (determined from filtered images). The depth of the pit can be followed for 9 A from the widest contour line. Steric limitations in imaging the cavity are due to the dimensions of the tip, and only the outer opening can be reliably measured. The largest circular contour line is taken as a reference for the outer opening and has a diameter, in this case, of 28-36 A (at the smallest and largest widths, respectively). The useful imaging time is limited by the rate of dehydration, since the average time for stability of the sample with respect to STM imaging was 60-90 min. After this length of time membranes underwent a substantial change in aggregation, resulting in smaller fragments moving freely under the probe. Such observation of a limited imaging time suggests a deleterious effect of dehydration on the sample. We therefore studied preparations of AChR-enriched membranes, treated as in the STM structural studies, with respect to their protein secondary structure by using CD spectroscopy and their functional stability by using 125I-a-bungarotoxin binding over the same time period. In Fig. 4 the CD spectra of membranes allowed to dry for two different times and then solubilized are presented and compared with a sample of the same preparation that was simply solubilized. Numerical values of the spectral data analyses are presented in Table 2. Only a slight difference in the intensity of the two troughs at 209 and 218 nm of the dried sample compared with the control preparation was detected. Comparison of curve 1 (control) and curve 2 (allowed to dry for 30 min) showed that there was a loss of 6% in helical structure and an increase of -4% in the random-coil signal. No significant differences were found between the samples dried for 30 min (curve 2) and for 3 hr (curve 3). However, as noted above, we did observe changes in the STM images over the longer time period. The binding of 125I-a-bungarotoxin to the AChR was not affected signif-
Proc. Natl. Acad. Sci. USA 89 (1992)
icantly by dehydration over the time periods used (180 min; Table 3). The slightly lower values of the dried samples (85 ± 11% of the control) may be due to small changes in structure of the protein or to incomplete solubilization of the AChR from the HOPG surface by the detergent.
DISCUSSION STM imaging of biological macromolecules has only recently been applied to nucleic acids (22-25), globular proteins (26, 27), and glycogen (28). A review of several such studies of proteins is presented in ref. 2. In addition, similar studies of membranes containing bacteriorhodopsin have been described (29). A very recent study of Torpedo marmorata membranes enriched in AChR showed arrays of receptor molecules rising above the membrane surface (30) with resolution comparable to negatively stained electron micrographs. DNA is a highly suitable substrate for STM imaging, presumably because of its characteristic shape and surface charge density. In the case of soluble proteins and glycogen, their overall shape and dimensions can be determined but few details of the surface have so far been determined, either because of a lower density of surface charge or a less characteristic shape compared with nucleic acids. In the study reported here the resolution obtained also does not reveal the structure at atomic resolution, but it has been possible to obtain overall dimensions for the outer (synaptic) surface of the AChR. The values obtained by using filtered images agree well with those previously reported from other methods. The characteristic shape and dimensions of the AChR, with a large central cavity, allowed facile identification of this membrane-embedded molecule. In this attempt to delineate the surface structure ofthis molecule by STM it was important to eliminate the possibility of artifactual images (31) due to factors such as the substrate used for sample deposition, the variability of the probes, or modification of the sample upon dehydration. Thus, comparison of the images and their dimensions with those previously obtained by other methods was critical (see ref. 10). Since no major changes in either secondary structure, as evidenced by CD spectroscopy, or in the extent of a-neurotoxin binding occurred over time up to 3 hr, it is likely that the loss of useful information after 60-90 min of imaging could be due to the effects of dehydration on the lipid bilayer. Related to this we found that detergent-solubilized AChR was less stable for imaging purposes than the membrane-bound form. In addition, the pH of the preparation affected the uniformity of its spreading on the graphite surface, which was much better under slightly alkaline conditions than at neutral pH. We observed similar effects of pH on the spreading of AChR samples on carbon-coated grids used for transmission electron microscopy, in that larger membrane aggregates were also observed at lower pH. The most revealing structural details ofthe AChR molecule are obtained by using unfiltered images where major regions of protein density are observed, two of which are somewhat overlapping (Fig. 3). These main regions of density may correspond to the constituent subunits of the AChR. Due to their disposition these features suggest a rather asymmetric structure, despite the known extensive homology in primary structure between the subunits (6, 7). We conclude that the STM method can at present be profitably utilized to obtain both surface dimensions and low-resolution structural information for membrane proteins when such membranes contain a high density of one specific protein such as the AChR or bacteriorhodopsin (29) or where two-dimensional ordering is observed in reconstituted systems where a highly purified membrane protein has been reintroduced into lipid bilayers. It is important to note that not only do the outer and inner dimensions of the AChR images reported here conform to the values obtained by other methods but also that the pattern of
Biophysics: Bertazzon et al.
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