Ultramicroscopy 42-44 (1992) 1194-1199 North-Holland

Imaging of subunit complexes of thermophilic bacterium H+-ATPase with scanning tunneling microscopy Junji Masai

a,

T e i k o S h i b a t a a, Y a s u o K a g a w a b a n d S h u n z o K o n d o c

Research Center, Mitsubishi Kasei Corporation, 1000 Kamoshida-cho, Midori-ku, Yokohama 227, Japan b Department of Biochemistry, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan c Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan

Received 12 August 1991

Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H+-ATPase of a thermophilic bacterium. The measurement was carried out in air without conductive coating on the samples deposited on a highly oriented pyrolytic graphite (HOPG). The F~ subunit complex of the H+-ATPase, and an H+-ATPase whose F~ portion was embedded into liposomes prepared from soybean lecithin were imaged. Overall structural images of the subunit complex Fa were obtained: the structural dimensions of the STM images are in agreement with those deduced from conventional methods such as an transmission electron microscopy (TEM) and small-angle X-ray scattering (SAX) experimentation. Regarding the STM imaging of these samples, we discuss the advantages and disadvantages of the STM over those of conventional methods such as a TEM and SAX.

I. Introduction T h e s c a n n i n g p r o b e microscope (SPM), used in s c a n n i n g t u n n e l i n g microscopy (STM) a n d atomic force microscopy ( A F M ) , has b e c o m e a useful tool for the real-space imaging of surfaces. T h e application of S P M to b i o m a t e r i a l s in ambient c o n d i t i o n s has growing potential. To date, a n u m b e r of biological materials have b e e n observed by SPM [1,2]: D N A ' s [3], D N A - p r o t e i n complex [4], polypeptide a n d p r o t e i n molecules [5], m a c r o m o l e c u l a r assemblies of p r o t e i n s such as flagella [6], m i c r o t u b u l e s [7] a n d actins [8], m e m b r a n e s a n d m e m b r a n e p r o t e i n s [9], a n d so on. I n this paper, we report the results of S T M imaging of r e c o n s t r u c t e d s u b u n i t structures of H + - A T P a s e . H + - A T P a s e (also called A T P synthase) is a reversible enzyme f o u n d in energyt r a n s d u c i n g m e m b r a n e s such as the i n n e r m e m b r a n e s of m i t o c h o n d r i a : it can either p u m p protons across the m e m b r a n e s against their electrochemical g r a d i e n t by hydrolysing A T P , or can

synthesize A T P w h e n p r o t o n s pass t h r o u g h the m e m b r a n e down the electrochemical p r o t o n gradient. H + - A T P a s e is also called FoF1-ATPase, b e c a u s e it is c o m p o s e d of a hydrophilic F~ sector a n d a hydrophobic F 0 one. T h e F~ sector is the ATP-catalytic part of the s u b u n i t complex, while F 0 is the p r o t o n - c o n d u c t i n g c h a n n e l of the complex. Fig. 1 shows a schematic drawing of F0F 1 a n d the stoichiometrical a3/33y8E s t r u c t u r e

F1

F0 Fig. 1. Schematic drawing of model structure of H+-ATPase whose F0 portions are embedded in a lipid bilayer.

0304-3991/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved

J. Masai et al. / Subunit complexes of H +-ATPase [10,11]. Presence of the TF l (thermophilic F 1 of TFoF1, TFoF 1 is FoF 1 of thermophilic bacterium PS3, the same applied to TF 0 and F 0) oligomer O~3fl 3 w a s demonstrated by biochemical methods such as p r o t o n - d e u t e r o n exchange kinetics [12] and gel-chromatography [13-15]. There has been much work done on the molecular shape of the F~ complex by using conventional methods such as transmission electron microscopy (TEM) and a small-angle X-ray scattering (SAX) technique. A hexagonal structure of T F 1 in two-dimensional crystals has been reported [16]. Computer processing [17] of the T E M images of the negatively stained crystals has shown their hexagonal structure. It has also been shown, based on SAX experiment [18], that the molecular shape of TF l is a hexagon composed of six identical ellipsoids. The conventional methods, however, are not necessarily advantageous in that they require expensive facilities and time-consuming analysis to obtain the images. However, an SPM technique is now available that is relatively efficient compared with conventional methods. The purpose of this paper is to report the STM images of the TF x subunit complex (molecular weight 385351) and a3/33 hexamer (major component of TF~, molecular weight 319582), and of the H+-ATPase whose F 0 portion was embedded into liposomes prepared from soybean lecithin, although the mechanism of STM imagings of these biomaterials is still unclear.

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STM was run in a constant-current mode. For the calibration of the STM image, see ref. [8]. 2.2. Sample preparation The subunits of TF 1 were overexpressed in E. coli (Escherichia coli) and were purified as reported by Ohta et al. [19]. TF 1 was purified as described by Kagawa and Yoshida [20]. Detailed descriptions of the sample preparation have been reported elsewhere [15,21]. In the STM experiment, a volume of approximately 5 /zl of diluted TF~ solution (approximately 6 / z g / m l ) was placed on a chip (5 m m × 5 m m × l mm) of freshly cleaved H O P G (highly oriented pyrolytic graphite) and dried for 1 night in air at room temperature. Similarly, approximately 5 tzl of a solution of TFoFl-liposome (a liposome in which the TF 0 portions of TFoF 1 were embedded) were spread on a similar H O P G chip and dried in the same way.

3. Results and discussion

3.1. Results 3.1.1. TEM image of TF1 The conventional T E M image of TF~ is shown in fig. 2 where the sample is negatively stained

2. Materials and methods

2.1. Scanning tunneling microscope STM experiments were carried out in atmospheric conditions with a commercially available STM (NanoScope II, Digital Instruments, Inc.) using a mechanically polished platinum-iridium (Pt-Ir) alloy (80%-20%, 0.25 mm diameter) tip. The STM measurements were performed at I t (tunnel c u r r e n t ) = 0.32 nA and Vt (bias voltage) = 358 mV for the T F t sample, and I t = 0.51 nA and Vt = 400 mV for the TFoF 1 sample whose TF o portions were embedded in liposomes. The tip scanning speeds were 0.69-3.5 H z / l i n e . The

Fig. 2. A high-magnificationTEM photograph of TFl; negatively stained with 1% uranyl acetate. Scale marker indicates 100 nm.

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with 1% uranyl acetate. In this image, it is difficult to see the individual subunit structures due to the resolution limit arising from the granule size effect of the negatively stained materials.

ened. The vertical range is 10 nm and the horizontal range approximately 10 nm. The hexagonal images in figs. 3a and 3b are thought to be the OL3~3 hexamer portion of the TF~ complexes.

3.1.2. STM image of TF1

3.1.3. STM image of the TFoFl-liposome

Fig. 3a shows a top-view STM image of T F t deposited on H O P G . T h e image is approximately 24 nm x 24 nm. In this image, as shown by the arrow (al), we can see hexagonal subunits with a concave structure in the centre of the complex which is similar to the a r r a n g e m e n t of the F 1 unit illustrated in fig. 1. T h e corrugation shown by a n o t h e r arrow (a2) is almost the same size, but is without clear concave structure. This is t h o u g h t to be an image of the T F 1 complex which might be located on the substrate in an upside-down fashion of the possible F~ complex structure shown in fig. 1. T h e other small corrugations are thought to be disassembled part of the TF~ complex (this point is discussed later). In figs. 3b, a perspective STM image of T F t after a smoothing treatment of original one is shown. T h e vertical scale is shortened and the horizontal one length-

A top-view S T M image of the TFoF~-liposome (liposome and T F o F 1 whose T F 0 portions are e m b e d d e d into the liposome) deposited on H O P G is shown in fig. 4. T h e image is 61 nm x 61 nm. A closer look at fig. 4 gives us m o r e information about the t o p o g r a p h y of the assemblies; we can see some corrugations (indicated by arrows in fig. 4) with concave structures in their centre. The corrugations are approximately 8 nm in diameter and hence are thought to be the TF~ portions of the T F o F ~whose T F 0 portions are e m b e d d e d into lipid bilayers of liposomes as illustrated in fig. 1. The resolution of this image is p o o r c o m p a r e d to those shown in figs. 3a and 3b, although the reason is as yet unclear. More experiments will be necessary to distinguish unambiguously these TFj portions of the T F o F ~ from the other corrugations seen in fig. 4.

Fig. 3. (a) An STM image of TFv Tunneling current, 0.32 nA; tunneling bias, 358 mV; scan area, 24 nmx24 nm. (b) A high-magnification STM image of TF1 after smoothing-treatment. The vertical scale is shortened and the horizontal one is lengthened. Scan area, 10 nm x 10 nm.

J. Masai et aL / Subunit complexes of H +-ATPase

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Fig. 4. A top-view STM image of TFl portions of the TFoF1 whose TFo portions are embedded in liposome. TF 1 portions are indicated by arrows. Tunneling current, 0.51 nA; tunneling bias, 400 mV; scan area, 61 nm x 61 nm. 3.2. Discussion

In general, there are some difficulties in the application of STM to biology: poor conductivity of biomaterials, flexibility of biomaterials and fixation of biomaterials onto substrates are major causes of decreased resolution and reproducibility of STM images. These difficulties also affect the present data: In fig. 3a, we can see some small corrugations which are smaller compared with the possible images of T F 1 shown by the arrows. The small corrugations are thought to be due to the following causes; during the drying process of the sample on H O P G , a certain amount of the T F 1 complexes was broken into smaller combinations of O/3fl3Tt~E a n d / o r individual units of O~3fl3Tt~E. In addition to this, during STM imaging, the scanning-tip could touch the sample and break the sample into smaller combinations of ot3fl3Tt~E because of a possibly poor conductivity. In fig. 4, the STM image is not clear for TF 1 portions of the TFoF 1 whose T F 0 portions are embedded into lipid bilayers of liposomes. This poor resolution might be due to a possible mechanism that tunneling through the lipid layer of liposome instead of tunneling directly into H O P G could reduce the contrast.

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As for the molecular shapes of the complexes shown above, there have been several reports: based on a small-angle X-ray scattering (SAX) experiment [18], it was shown that the molecular shape of T F 1 is a hexagon composed of six identical ellipsoids (a = b = 1.85 nm and c = 7.4 nm) arranged around a smaller ellipsoid (a = b = 2.8 nm and c = 4.5 nm). In addition, SAX data [22] revealed that the size of a certain isolated subunit from F 1 was 4.5 × 4.5 x 10.5 nm, in the estimated form of an elliptic cylinder. The recent p a p e r by H a r a d a et al. [15] also reported an SAX experiment that showed the radius of gyration (Rg) of the O~3~3 hexamer to be 4.64 nm. As far as the T E M observation of H+-ATPase is concerned, the hexagonal structures of the two-dimensional crystal of the T F l complex were reported by Wakabayashi et al. [16] and Yoshimura et al. [17], respectively. More recently, Gogol et al. [23] showed ligand-dependent structural variations in EF 1 (F l subunit of H+-ATPase of E. coil) H ÷ATPase by using cryoelectron microscopy. Our present images of TF~ complexes are consistent with the results of the reports explained above. The previously mentioned conventional methods such as a computer processing of T E M photographs and SAX experiments, however, have disadvantages; these require expensive facilities and time-consuming analysis to obtain the images. However SPM is now available that can process data almost immediately using relatively inexpensive and compact equipment. From the viewpoint of sample preparations of biomaterials and of the precise determination of sample structure, both conventional methods and STM have assets and drawbacks. Which method is preferential seems to depend on the nature of the sample and the sample preparation procedure used in the observation. If we could obtain enough pure solution or enough cubic crystal of a biomacromolecule, it would be convenient to use X-ray crystallography or an SAX experiment so that the structural information obtained would be more precise than an STM image could provide. However, if we could not obtain crystals, it would be convenient to use STM so that an image of a single molecule could be obtained. Meanwhile, in the case of m e m b r a n e - b o u n d biomaterials such

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as receptors and channel-forming substances which are embedded in cell membranes or liposomes, the conventional methods might not be preferable; it seems that X-ray methods are almost impossible and computer-processing methods for TEM images are not applicable unless the biomaterials form symmetric arrangements in the membranes. On the other hand, with the use of STM, it is expected to observe membrane-bound biomaterials. In fig. 4, we have shown a preliminary result suggesting the possible existence of TF 1 portions of the TFoF 1 whose TF 0 portions are embedded into lipid bilayers of liposomes. As for the continued study of imaging of biological materials with STM, one of the possible key techniques to be developed in the imaging of protein subunit assemblies would be metal-labelling of the specific subunit in combination with a possible antigen-antibody reaction [24]. This technique was used to clarify the binding site of IgG antibody complex by Olk et al. [25], who followed our study on the preliminary experimentation using an STM immunoassay [24]. STM imaging of a membrane-bound protein labeled with a metal was also reported by Horber et al. They showed an STM image of a cell membrane containing concanavalin-A labeled with 20 nm colloidal gold particles [26]. Therefore, we expect the STM immunoassay technique to be useful when we try to image membrane-bound proteins such as acetylcholine receptor and the present sample of TFoF ~ embedded in liposome. The possibility may exist that the specific binding site of ligands and membrane-bound proteins could be identified by using small metal particles such as gold colloids and the immunoassay technique. We are now planning to image receptor molecules embedded in liposome using the STM immunoassay technique proposed previously.

4. Conclusions The overall structural images of the TF 1 subunit complexes of the thermophilic bacterium H+-ATPase were obtained using a scanning tunneling microscope. The structural dimensions of the STM images are in agreement with those

deduced from conventional transmission electron microscopy and small-angle X-ray scattering experiments. Regarding the STM imaging of these samples, we discussed the advantages and drawbacks of STM in comparison with conventional methods such as T E M and SAX experiments. The present measurements of the subunit complexes suggest that STM is potentially useful to image H+-ATPase and other individual macromolecular structures of biological importance composed of self-assembled subunits.

Acknowledgements This work was performed under the management of the Research Association of Biotechnoiogy, a part of R & D Basic Technology for Future Industries, supported by N E D O (New Energy and Industrial Development Organization). Thanks are also due to Mr. Gen Nomoto, Mr. Bunpei Sato and Dr. Kenji Nagahari for their support of this research project.

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Imaging of subunit complexes of thermophilic bacterium H(+)-ATPase with scanning tunneling microscopy.

Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H(+)-ATPase of a thermophilic bacterium. The measurement w...
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