J. Phys. Chem. 1993,97, 8852-8854
8852
Studies of Covalently Immobilized Protein Molecules by Scanning Tunneling Microscopy: The Role of Water in Image Contrast Formation Graham J. Leggett, Martyn C. Davies, David E. Jackson, Clive J. Roberts, Saul J. B. Tendler, and Philip M. Williams The VG SPM Laboratory for Biological Applications, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK Received: June 7 , 1993@
Protein molecules have been immobilized on gold surfaces using a novel covalent attachment technique. The naked molecules have been imaged with the scanning tunneling microscope (STM), and the effects of protein dehydration on S T M image contrast have been investigated. A reversal of image contrast is observed upon reduction of the relative humidity inside the S T M chamber from 33% to 5%. It is concluded that water plays a crucial role in the formation of the S T M image of the protein molecules under ambient conditions.
Studies of biomolecular structure and function by scanning tunneling microscopy (STM) are beset by three critical problems:1*2 molecular movement caused by tipsample interactions, the observation of substrate features which mimic biomolecular structures, and the lack of an adequately confirmed model to explain contrast formation in the STM image. The development of appropriate methods of sample preparation is an essential precondition to the solution of these problems. There are three principal approaches: first, application of a physically robust conducting~ o a t i n g ;second, ~ . ~ electrochemical immobilizationof biomolecules;5 third, the introduction of a covalent bond between the biomolecule and the surface.6 The latter two approaches are preferable in that they facilitate the imaging of naked molecules: if sufficiently stable arrays of molecules could be formed, it would become possible to examine their responses to physical and chemical stimuli using the STM. The only other realistic alternative is the examination of two-dimensional arrays (for example, two-dimensional crystals') where biomolecules exhibit a tendency toward spontaneousself-organization. In the present study we examine the effect of dehydration on covalently immobilized catalase molecules. The results have a strong bearing on the debate concerning the nature of the image formation mechanismin STM. A wide variety of models have been proposed to account for the fact that it is possible to image protein molecules with the STM. Some have suggested that deformation of the adsorbate by the STM tip alters its electronic structure such that resonant tunnelingcan occur. Competingtheories have suggested that water plays an important role, by providing a conducting path. The hypothesis that water is involved in the formation of the STM image may be tested straightforwardly by examining the effects on image contrast of dehydration of the biomolecules. Gold surfaces were prepared by annealing gold wire (0.5" diameter; Johnson-Matthey, Royston, Herts, UK) in a bunsen flame. Melting of the gold wire resulted in the formation of a (1 11)-faceted ball of diameter 1-2 mm. The formation of Au(1 11) facets in this fashion has been widely reported;899the facets may be atomically flat over distances up to hundreds of nanometer^.^ A gold ball was inserted into a 1mMol dm-3 solution of 3-mecaptopropionic acid (MPA; Sigma, Poole, Dorset, UK) in deionized water for 30 min. The MPA chemisorbs onto the gold surface1&12forming an ordered monolayer, with the MPA sulfur atom binding to the gold substrate and the carboxylic acid group free. Following removal of the gold ball from the MPA solution, it was rinsed in flowing deionized water for 10 s. The gold ball was then activated by insertion into a 40 mMol dm-3 solution of 1-ethyl-3- (3-(dimethylamino)propy1)carbodiimide e Abstract
published in Advance ACS Abstracts, August 15, 1993.
0022-3654/93/2097-8852$04.00/0
Figure 1. Clean Au( 111) facet, exhibiting atomically flat terraces. Image size: 389 X 389 nm.
(EDC; Sigma), in deionized water at pH 4.7, for 5 min. The final step was performed by transfer of the gold ball immediately, and without washing, to a solution of the protein (Sigma), at a concentrationof 3 pg/mL and pH 7.8, for 16h. Following removal of the gold ball from the protein solution, it was rinsed in flowing deionized water for 10 s. The STM studies were performed on a VG STM 2000 system (VG Microtech, Uckfield, Sussex, UK). Except where otherwise stated, the instrument was operated in constant current mode with a sample bias voltage of +1.6 V and a tunnel current setpoint of 10-20 PA. Platinum/iridium tips were prepared mechanically from Pt/Ir wire (80/20; Agar Scientific, Stansted, Essex, UK). The humidity inside the STM chamber was measured using a Vaisala HMI 31 Humidity Meter (RS Components,Corby, Northants, UK), with the probe of the meter placed close to the sample. Silica desiccant (Englehard Corp., Cleveland, OH) was activated by baking at 140 "C for 16 h. A suitable area of the sample was located using the STM. The STM tip was then retracted (by ca. 2 pm), and the desiccant inserted. The STM chamber was sealed following insertion of the dessicant (the VG 2000 system is designed to UHV specifications,although ours is not equipped with a UHV pumping system). The tip was brought back down and a fresh image recorded. Provided care was excercised, the STM tip could be brought down to within 100 nm of its original position. 0 1993 American Chemical Society
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The Journal of Physical Chemistry, Vol. 97, No. 35, 1993 8853
Figure 2. Islands of immobilized protein molecules. (a) Image size 295
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The surfaces of the clean gold facets exhibited atomatically flat regions extending over hundreds of nanometers, exhibiting monatomic steps and terraces (Figure 1). The STM images of gold surfaces to which catalase had been covalently immobilised were dominated by large island structures some 5-10 nm in height (Figure 2a). All of these islands were distinguished from the surrounding substrate by a much rougher surface topography. This rough, or mottled appearance was due to the presence of the immobilized protein molecules within the island regions. This becomes clear in the high resolution image shown in Figure 2b. The globular catalase molecules are seen to be closely packed in a dense array. As a consequenceof the closeness of the packing, it is difficult to determine the dimensions of the molecules accurately, although an estimate of the molecular diameter would be in the region 7-8 nm. This figure is in reasonable agreement with figures obtained by electron microscopy:137.5 and 9 nm in the x and y directions (respectively). Validation of this interpretation was achieved straightforwardly by examining the effect of reversal of the samplebias potential.6 Under normal conditions, the sample bias voltage is positive. With a negative bias voltage, regions of the gold substrate retained essentially unchanged contrast, whereas the island regions exhibited a marked change in contrast. This differential response strongly supports our interpretation that the island features are formed from immobilized protein molecules. A detailed discussion of relevant aspects of sample preparation has been provided elsewhere.6 Figure 3a shows an image recorded under ambient conditions, with a relative humidity of 33%. A number of island regions are observed. The island regions are observedas having a light (high) contrast, and the substrate is observed as having a dark (low) contrast. We draw attention to one in particular which is distinguished by a sharp notch in its side (indicated by an arrow in the figure). The desiccant was inserted into the STM chamber which was then sealed. Images were recorded as the humidity inside the STM chamber decreased. Images were recorded over some 18 h, and, during this period, there was some gradual drift in the position of the STM tip. However, the movement was only slight, as can be seen from the small change in the position of the arrow in the sequence of images. Such was the stability of the island structures that repeated cycles of dehydration and rehydration could be performed on the same area of the sample with essentially little structural change (under ambient conditions, identical images were repeatedly obtained after each cycle), although variations were observed in the image contrast as the humidity changed inside the STM system. After desiccation for 2 h, the relative humidity had reduced to 10% and the image had become complex (Figure 3b): the
295 nm. (b) Image size 68
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distinction between the island regions and the substrate had become less clear because of a general darkening of the island regions corresponding to a decrease in the observed height. However, after 16 h of desiccation and a reduction of the relative humidity to 5%, there had been a complete reversal in the image contrast (Figure 3c). The island structures were then observed to have low (dark) contrast, whereas the substrate was now observed to have high (light) contrast. Removal of the desiccant, and exposureof the sample to ambient conditions,rapidly restored the original image contrast (Figure 3d). To summarize: under normal (hydrated) conditions, and in constant current mode, the protein islands are observed to be higher than the gold substrate; under dehydrated conditions, the protein islands are observed to be lower than the gold substrate. Under hydrated conditions, the protein molecules conduct and so, in order to maintain a constant tunnel current, the tip is raised as its traverses the island of protein molecules. The dehydrated protein molecules conduct much less well, however, and as the tip traverses the dehydrated protein island it has to be lowered to maintain a constant tunnel current. Finally, we investigated the effects of dehydration on features of the gold substrate. Irregular structures not dissimilar to those obtained using the covalent coupling procedure were obtained on occasions, and images were recorded sequentially as the relative humidity inside the STM system was reduced to 5%. Whereas the contrast of the image of the protein island was markedly altered under these conditions, the gold substrate feature retained unaltered topography (data not shown). Thus an additional consequenceof the differential response of the gold substrate and the protein island structures to changes in hydration is that a further means is provided for distinguishingbiomolecularmaterial from substrate features. Protein molecules have little intrinsic conductivity, and it is therefore remarkable that STM images of proteins may be recorded at all. There has consequently been considerabledebate about the mechanism of STM image formation for protein samples. However, there has been, to date, no conclusive empirical evidence that water is involved in the formation of the STM image. Guckenberger et aL7 have suggested that it is difficult to obtain images at relative humidities below 30%, implying that hydration of thick biological samples is a prerequisite for the recording of an STM image. Our data provide unambiguous support. Of particular importance is the reversal of contrast observed at very low relative humidity values. It is clear therefore that water plays an essential role in the formation of the STM image under ambient conditions. Careful consideration of environmental factors, and in particular the relative humidity, is
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8854 The Journal of Physical Chemistry, Vol. 97, No. 35, 1993
a
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d
Figure 3. Effect of dehydration on image contrast. Relative humidity values: (a) 33%, (b) lo%, (c) 5%, and (d) 33%. Image size 413 X 413 nm.
essentialfor the full exploitationof STM in studiesof biomolecular structure and function. Acknowledgment. The financial support of the SERC/DTI
Protein Engineering Link Scheme, Glaxo Group Research and VG Microtech is gratefully appreciated. References and Notes (.l) Salmeron, M.;Beebe, T. P.; Odriozola, J.; Wilson, T.; Ogletree, D. F.; Siekhaus, W. J. Yac. Sci. Technol. 1990, A8, 635. (2) Clemmer, C. R.; Beebe, T. P., Jr. Science 1991, 251, 640.
(3) Reneker, D. H.; Schneir, J.; Howell, B.; Harary, H. Polym. Commun. 1990.32, 167.
(4) Leggett, G. J.; Wilkins, M. J.; Davies, M. C.; Jackson, D. E.;Roberts, C. J.; Tendler, S . J. B. Langmuir 1993, 9, 1115.
( 5 ) Lindsay, S. M.; Tao, N. J.; DeRose, J. A.; Oden, P. I.; Lyubchenko,
Y.L.; Harrington, R. E.; Shlyakhtenko, L. Biophys. J. 1992, 62, 1570. (6) Leggett,G. J.; Roberts, C. J.; Williams, P. M.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. Langmuir, in press. (7) Guckenberger, R.; Wiegrabe, W.; Hillebrand, A.; Hartmann, T.; Wang, Z.; Baumeister, W. Ultramicroscopy 1989, 31, 327. (8) Schneir, J.; Sonnenfeld, R.; Marti, 0.; Hansma, P. K.;Demuth, J. E.;Hamers, R. J. J. Appl. Phys. 1988, 63, 717. (9) Clemmer, C. R.; Beebe, T. P., Jr. Scan. Microsc. 1992, 6, 319. (10) Ihs, A.; Liedberg, B. J. Colloid Interface Sci. 1992, 244, 282. (1 1) Uvdal, K.; Bodo, P.; Liedberg, B. J. Colloid InterfaceSci. 1992,249, 162. (12) Leggett, G. J.; Davies, M. C.; Jackson, D. E.;Tendler, S.J. B. J. Chem. SOC.,Faraday Trans. 1993,89, 179. (13) Furuno, T.; Ulmer, K.; Sasabe, H. Microsc. Res. Technol. 1992,21, 32.
