J. Phys. Chem. 1995,99, 16155-16161
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Effect of Controlled Hydration on Scanning Tunneling Microscopy Images of Covalently Immobilized Proteins Marie-Claire Parker, Martyn C. Davies,*J and Saul J. B. Tendler*i' Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, Nottingham NG7 2RD, U.K. Received: March 17, 1995; In Final Form: August IO, 1995@
The enzyme catalase has been covalently bound to a self-assembled monolayer adsorbed to a gold substrate using a novel, organic solvent-based, attachment method. The enzyme was shown to remain catalytically active after immobilization to the surface. Under conditions of controlled humidity the effect of protein hydration on the scanning tunneling microscopy images of the naked molecules were investigated. Data were collected over a range of hydration conditions, from near zero to 86% relative humidity. Significant and reversible changes in the image quality were observed when the protein was dehydrated and hydrated relative to ambient humidity conditions. The findings are discussed in terms of the physical properties of hydrated proteins within the STM experiment.
1. Introduction The advent of scanning probe microscopes (SPMs) has provided a new range of tools for studying biological material immobilized at surfaces.'-3 Scanning tunneling microscopy (STM)has been used to study immobilized biomolecules in aqueous s o l ~ t i o nand ~ ~under ~ ambient air conditions.6-s However, the investigation of structural features by STM at the molecular level and the effect of physicochemical changes on the protein image has to date been limited. Recognized problems of image validation have arisen due to probe convolutiong and also from the widely reported artifacts observed on the substrates commonly employed for the imaging of biomolecules, specifically goldlo and Often, features observed can be virtually identical to the predicted topography of the target biological molecule, and therefore, the importance of image validation under such conditions becomes crucial. One approach used to indicate the presence of a biological molecule has been to attempt to move it under the sweeping force of the probe tip.I3 Generally, physically adsorbed molecules can be swept from the substrate by decreasing the gap resistance whilst a degradation to image quality can normally be observed for more rigidly anchored molecules, for example those covalently bound. A change to the tip bias from positive to negative can also result in a change to the image quality of the bound biomolecule.6 A method employed by Leggett et al.7 to aid in distinguishing between substrate artifacts and immobilized biomolecules on gold was to dehydrate the sample containing the introduced biomolecule. It was found that regions containing immobilized protein showed a reversal in image contrast upon dehydration from ambient humidity (33%) to dry conditions (5%). Regions where no molecules could be observed showed no such change to image contrast. To ensure routine reproducible imaging of naked biomolecules, a predictable and stable coverage, preferably on an atomically flat surface, is required. It has been shown that biomolecules can be immobilized to the surface of materials such as g ~ l d , ' ~~,i'l~i c o n , ' ~or . ' ~indium-tin oxideI8 for subset Telephone Number: 44-(0)115 9515063. Fax Number: 44-(0)115 95 15 110. E-mail Address:
[email protected]. E-mail Address:
[email protected]. Abstract published in Advance ACS Abstracts, September 15, 1995.
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quent SPM imaging. The method employed should provide rigid anchoring under nondenaturing conditions. A number of approaches to the covalent attachment of biomolecules to surfaces have been developed. Several techniques exploit the strength of thiol adsorption onto gold. Firstly, the protein may be modified6 to introduce the appropriate functionality to immobilize the biomolecule to the surface. Alternatively, the substrate surface may be modified with a bridging molecule that binds the p r ~ t e i n . ~ , 'A~ large , ~ ~ range . ~ ~ of thiol-containing molecules are known to spontaneously adsorb from solution onto gold surfaces, forming stable, well-ordered self-assembled monolayer^'^.^^ (SAMs). The adsorption of both thiols and disulfides is thought to result in the formation of the same chemical species, i.e. a gold t h i ~ l a t e . ~ ' The principal prerequisite for STM is that the sample should be sufficiently conducting to provide a tunneling pathway. The electrical conductivities of many biomolecules have been measured49(normally using compressed powder samples). Such measurements have shown that they can exhibit very high resistivities (of the order of 10'5-10's 52 m at room temperature). The mechanism, therefore, by which S T M images of protein can be obtained is a matter of considerable interest. A number of models based upon electron-tunneling mechanisms have been proposed to explain the contrast in such i m a g e ~ , ~ I - * ~ although there is no generally accepted mechanism whereby electrons can tunnel through an insulator. Yuan et have suggested that when samples are covered with a thin film of water the measured current is carried by ions rather than tunneling electrons and that both temperature and humidity play a crucial role. Recently, STM images of DNA on mica were obtained by scanning at very low currents ( < l PA). In that work and in others, a dependence in the STM images on the water content of the sample was ~bserved.',~ Although proteins may be classed as electronic insulators, they are able to display electr0nic,4~ionic:0 and protonic c o n d ~ c t i o n . ~ ~ Specifically, , ~ ~ , ~ ~ , ~ proteins I upon hydration have been shown to exhibit increasing protonic conduction.26-28 Protonic conductivity has been measured as a function of hydration24for proteins and biosystems such as maize seed26 and Artemia In all cases, as the systems were hydrated, a percolation threshold was observed, and above this, the conductivity showed an apparently exponential dependence on
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hydration. For the enzyme lysozyme,28there was approximately a four hundred fold increase in dc conductivity when it was hydrated from a water content of 0.15 g of water per gram of protein to one of 0.35g/g. The protonic conductivity displayed percolative behavior and appeared to reflect an extended network of water molecules bound on the surface of the proteins. The precolative threshold was found to coincide with the onset of biological function in the proteins studied.26 Clearly, changes to protein conductivity as a consequence of hydration are interesting from a practical standpoint as well as from a mechanistic one. Thus, an understanding of the specific molecuar events that are central to characterizing protein hydration is essential if the contribution hydration makes to STM image contrast formation is to be assessed. Here we report an STM study of protein molecules immobilized via a self-assembled monolayer to a gold surface using a novel, organic solvent-based approach. The molecules have been imaged under different conditions of controlled protein hydration. This study is particularly relevant, since following immobilization the enzyme catalase was shown to be catalytically active. Changes to the STM image contrast observed upon altering the water content of the protein will be discussed.
2. Experimental Section A. Preparation of Gold Substrates. Gold wire (JohnsonMatthey, Royston, Herts, U.K.) was immersed for cleaning purposes in a piranha solution (7:3concentrated sulfuric acid 30 wt % hydrogen peroxide), and the solution was stirred gently for about 1 min. The wire was removed and rinsed exhaustively in deionized water (resistivity, 18 MQ cm, obtained from a Millipore filtration system) and then melted in a bunsen burner and allowed to quench in air. The melting of the wire results in the formation of a Au( 111) faceted ball of typically 2 mm in diameter. After the gold ball was quenched in air, it was inserted into the piranha solution in order to remove any organic contaminants formed on the surface as a result of the flame annealing process. The gold ball was then rinsed exhaustively in flowing deionized water. Excess water was removed from the gold ball via capillary action using filter paper. The ball was then allowed to air dry. B. Preparation of Protein. Catalase (E.C. 1.11.1.6) from bovine liver was received as a crystalline powder (Sigma, Poole, Dorset, U.K.). The protein was dissolved in sodium phosphate buffer (10 mM, pH 7.8)to a concentration of 5 mg/mL. The solution was flow-dialyzed against deionized water at 4 "C and lyophilized. The resulting freeze-dried powder was then stored in a sealed containiner over 3 A molecular sieves. C. Covalent Coupling in Anhydrous Organic Solvent. The coupling reagent, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Pierce, Luton, U.K.), was dissolved in anhydrous 2,2,2-trifluoroethanol(TFE) (99%+,Aldrich, Gillingham, Dorset, U.K.) to a concentration of 0.95 mM. A freshly prepared gold ball was suspended in this solution using clampable tweezers for a period of up to 5 h, removed, and transferred to a solution containing approximately 0.4 pM catalase in TFE. A similar volume of N,N-diisopropylethylamine (DIPEA, 99.5%, Aldrich, Dorset, U.K.) was then added. The gold ball was incubated in this solution for approximately 21 h and washed exhaustively in flowing deionized water in order to remove any noncovalently bound protein. STM imaging of the immobilized protein was carried out within 48 h. D. Hydration of Covalently Immobilized Catalase. The samples were hydrated in the STM chamber. The humidity was
Parker et al. controlled by placing two petri dishes containing the desired saturated salt of known vapor pressure28 inside the STM chamber, which was then sealed and allowed to equilibrate to the desired humidity for a period of approximately 2 h before any STM images were taken. This period of time was necessary to ensure that protein hydration had reached equilibrium, a process that is normally dependent on the quantity of p r ~ t e i n . ~ ~ . ~ ~ The humidity of the chamber was routinely checked using a humidity meter (Vaisala HMI 31, R.S. Components, Corby, Northants, U.K.) placed inside the chamber. The air temperature remained constant throughout (by means of air conditioning) at 20-21 "C. Inorganic salts (Aldrich) were used as supplied. The corresponding relative humidity values (RH) at 20 "C associated with the water-saturated salts are listed here: lithium chloride (11%); potassium acetate (22%); potassium carbonate (43%); sodium bromide (58%); potassium iodide (69%);sodium chloride (76%); potassium chloride (86%). A mixture of freshly regenerated 3 A molecular sieves and silica gel was used to dehydrate the sample in the STM chamber. E. Assessment of Catalytic Activity of Catalase after Immobilization to Gold. Glass microscope slides were cut into sizes of approximately 3.75 cm2. The slides were then coated with dimethyldichlorosilane by immersing them in a stirring 50 mM solution. They were then removed, rinsed exhaustively in flowing distilled water, and allowed to air dry. Sputter coating with gold was achieved in a SC 510 coater (VG Microtech, UcWield, U.K.) operated at 0.1 mbar, with a sputtering current of 30 mA and a coating time of 4-6 min. The gold slides were treated with the disulfide coupling reagent and then exposed to the catalase solution in the same manner as described above. Noncovalently bound protein was removed by rinsing exhaustively in deionized water. Gold slides with covalently immobilized protein were immersed in a vial containing a solution of 0.8 mM hydrogen peroxide (Aldrich) and a few crystals of sodium iodide. The vial was shaken gently in order to cover the slides with solution, and after a few minutes a drop of starch indicator solution (1 wt % solution in water, Aldrich) was added. Starch indicator was used to test for the presence of iodine formed by the reaction of undecomposed hydrogen peroxide. In all assessments of catalase activity, no color change was detected, indicating that all the hydrogen peroxide had been decomposed by the active, immobilized catalase. This test was performed repeatedly on the same slide for at least three washings and showed the same effect in each instance. Controls containing gold-coated slides with no protein present were simultaneously performed. A change of the solution color to blueblack on addition of the indicator was detected in all control reactions. It was determined that 0.24 pM of hydrogen peroxide was the minimum quantity necessary for a visible color change upon addition of starch indicator in the control reaction. To ensure that catalase immobilized to the gold ball was active, an assay was also performed using one of the gold balls prepared with immobilized protein. The gold ball with immobilized protein was immersed in the stirring solution and left for a period of 10 min. A control vial, free of catalase was treated in exactly the same way. After this time a spatula tip of sodium iodide was added to both vials followed by starch indicator. In the control vial a faint blue color was immediately detected; in the vial containing the gold ball, no color change was apparent. The UV absorbance for the decomposition of hydrogen peroxide (200 mM) by catalase was measured at 240 nm; the concentration of catalase was 10 nM. The decomposition of hydrogen peroxide (3 mM) by catalase was measured at 240 nm.
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STM Images of Covalently Immobilized Proteins
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Figure 1. Schematic representation of protein coupling to the selfassembled monolayer of SPDP on gold.
F. Catalase Hydration Isotherm. Approximately 20 mg portions of dialyzed, lyophilized, and dried catalase were weighed into glass vials. The vials were then placed in sealed containers with the appropriate saturated salt and allowed to equilibrate at room temperature (20 Z!C 1 "C) for a period of 48 h. After this time, protein-water equilibration was assumed to have occurred and the vials were then resealed and reweighed. Samples were weighed to an accuracy of 0.1 mg (Sauter D-4740, Ebinger, West-Germany). The amount of water adsorbed by the protein was estimated from the measured change in weight. G. Instrumentation. Scanning tunneling microscopy studies were performed on a VG 2000 STM system (VG Microtech Ltd., Uckfield, Sussex, U.K.). Constant-current mode was employed throughout, with a sample bias voltage of typically f l V and a tunneling current set point of 5-10 PA. Platinum/ iridium (80:20, Johnson-Matthey, Royston, Herts, U.K.) tips were prepared by mechanically cutting a length of wire 0.5 mm in diameter. 3. Results and Discussion
A. Coupling of Protein to a Self-Assembled Monolayer. A reaction scheme detailing the novel procedure we have employed to prepare the covalently immobilized catalase is shown in Figure 1. N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) is a bifunctional cleavable crosslinker, containing an N-hydroxysuccinimide ester group and a 2-pyridyl disulfide group. The hydroxysuccinimide ester reacts with primary amino groups to give a stable amide bond, and the reaction proceeds rapidly under very mild conditions in aqueous media. In these conditions, however, it was found that the N-hydroxysuccinimide ester was rapidly hydrolyzed; the half-life for the ester
(23 "C) in water at pH 9 was approximately 1 min, and at pH 7.5, the half-life was 14 min, as observed by a W assay (data not shown). Rapid hydrolysis of the ester leads to a poor degree of amide bond formation and hence a low level of protein surface coverage. One possible means of avoiding ester hydrolysis is to use a dry organic solvent as the medium in which to carry out the coupling reaction. The absence of water in the solvent will result in the reaction equilibrium favoring synthesis over hydrolysis. The rate of decomposition of the ester in dry organic solvent was found to be very much lower than that in water. For example, SPDP dissolved in anhydrous trifluoroethanol typically retained > 80% of its ester content for periods of up to 3 weeks at 21 "C (data not shown). These findings were similar to those previously reported3*for SPDP dissolved in 99.5% ethanol. Protein coupling in an organic solvent is a novel method for preparing proteins immobilized at surfaces, and in choosing an organic 'solvent for this purpose, there are several main considerations: Proteins are insoluble in most organic solvents and, in some, they can denature.33 Therefore, a dry organic solvent which stabilizes the protein's secondary structure and has sufficient polarity to solvate the protein is required. 2,2,2Trifluoroethanol is known to successfully fulfill both criteria. The method that we have developed is the first example of an organic solvent-based coupling method which provides the opportunity to couple proteins to surfaces where the effect of competing hydrolysis is obviated. Organic solvents are known to dissolve small amounts of protein,33 and the retention of enzyme catalytic activity in these solvents has been well d ~ c u m e n t e d . ~ ~For - ~ ' example, solid-state NMR studies37have shown that the integrity of the active site of a protein (a-lytic protease) was retained in a number of organic solvents. Therefore, using a dry polar protic solvent that results in retention of catalytic performance may be considered a valid approach for the preparation of immobilized protein. B. Assessment of Catalytic Performance of Catalase after Coupling to Gold Surfaces. Catalase has an extremely high catalytic efficiency (kl = 4 x lo7M-' s - ' ) , ~ and ~ a colorimetric assay was developed that could rapidly determine if catalase remained active after coupling to gold surfaces. Undecomposed hydrogen peroxide reduces the added sodium iodide crystals to iodine, which produces a color change upon addition of starch indicator. After coupling the protein to the gold ball, the activity of catalase was assessed and catalase was found to have remained catalytically active. Since catalase was found to be active after coupling to gold from the TFEDIPEA solvent system, it could be assumed that TFE did not have any significant adverse effects on the enzyme. Absolute catalytic rates could not be measured because of the difficulty in determining the level of immobilized enzyme. A comparison between catalase concentration in aqueous media and catalase dissolved in TFEi was made. It was found that there was a 1015% decrease in the relative concentration for enzyme redissolved back into aqueous media. In our experiments, protein coupling to SPDP in trifluoroethanol was allowed to proceed by the addition of a hindered tertiary nucleophilic base, N,N-diisopropylethylamine (pKa 11). The addition of this organic base was necessary in order to deprotonate lysine residues on the surface of catalase, allowing covalent coupling to the S A M of SPDP. No catalase could be detected by STM if this base was not present in the solution. This observation is a further indication that the protein molecules we have observed are covalently bound to the SAM and not physically adsorbed to the gold surface. This finding
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Figure 2. 5.151 ~ i n a g c01 ii cleiiii Aui I I I )l'xel, exhibiting atomically flat v r r ~ c e \ . Imagc iix is 497 nrn x 497 nm.
correlates well with the literature, where DIPEA has been used in a similar capacity for conventional peptide syn~hesis?~ C. Imaging of Bound Catalase. In order to remove protein buffer salts for these experiments, catalase was dialyzed exhaustively against deionized water. The reasons were twofold firstly, reports have speculated that the presence of proteinbound salt can result in ill-resolved images, with image definition at molecular resolution being unattainable: secondly, in order to fully investigate the role of water in contrast formation, it was necessary to ensure that a large percentage of bound ions were removed. Ion conduction in proteins has been postulated as a mechanism of image contrast f ~ r m a t i o n .By ~~ minimizing the presence of ions, the effect of ion conduction pathways contributing to changes in contrast is significantly reduced. Figure 2 shows a scanning tunneling microscopy image of a bare Au(l1 I ) facet, typical of many observed. Atomically flat regions extending over several hundred nanometers, monoatomic steps, and terraces can be clearly seen. Initially, different regions of the gold substrate with the catalase covalently bound to the SAM were imaged and an appropriate area, most often atomically flat, was identified for subsequent scanning under controlled humidity conditions. Typically, large scan areas (200 nm x 200 nm) were recorded on an area containing distinctive features. The tip was lowered and raised repeatedly to ensure that it could be brought down close to its previous position. This was found to be the case and was typically less than 100 nm from its starting point. We have previously shown6 that the dimensions of catalase image by STM compare favorably with those determined from X-ray crystallographic (10.5 nm x 10.5 nm x 6 nm) data." Catalase is composed of four identical heme-containing subunits, each of a molecular mass of 60 kDa. The molecule is subdivided into four domains by two orthogonal grooves. one of which is deeper. In Figure 3 STM images of covalently immobilized catalase are shown, indicating the effect of STM image contrast of the amount of protein-bound water. The images show a near continuous layer of catalase with pin holes visible in the bare gold substrate and are typical of what is commonly observed in this laboratory. Due to close packing on the surface, the images do not show individual protein molecules and it was
Parker et al. not possible to obtain images showing protein structure at a molecular level. Higher resolution imaging did not reveal any further srmctural information other than that obtained at the image resolutions given in the data set. In Figure 3a the reduction to image contrast compared to that of the bare gold substrate for a sample dehydrated to a water content of near zero is apparent, the images appear to be very similar to that of a bare gold substrate. The contrast is only slightly enhanced as the humidity is increased to 22% RH (Figure 3b). However, when the same sample was hydrated to 43% RH (Figure 3c), a significant enhancement to the contrast was observed. This change continued as the sample was hydrated beyond this level to 58%. 69%. and finally a hydration level of 86%. as shown in parts d, e, and f, respectively. Beyond a hydration level of 69% RH, streaking of the image occurred. The changes to image contrast that we have observed are reproducible and also reversible. By altering the humidity of the chamber whilst imaging the same area, these changes to image contrast can be clearly observed. In the control experiments carried out, both the bare gold surface and gold containing the bound SAMs showed no change in STM contrast upon changing the humidity. Figure 4 shows the amplitude of the corrugation within the image as a function of relative humidity. The data shown have been taken from successive scan measurements on the same area as a result of increasing or decreasing the relative humidity. The results clearly suggest an increase in surface roughness with increasing hydration. At low humidity ((22% RH) there is a small change to the measured z-height for a 20% increase in relative humidity from near zero. Thereafter, the increase in corrugation continues throughout the humidity range toward a plateau level at 86% RH, suggesting that as the protein is hydrated, the conductivity of the sample increases. Surface roughness values were (root mean square, standard deviation of the z-values) measured on several areas at different humidity levels; these vary from 0.14 nm at near 0% RH to 0.42 at 86% RH. Apparent z-heights can however be variable when imaged over several different areas containing immobilized protein and may reflect differences in protein packing over the surface. Interestingly, such changes in image contrast are not seen for features on clean gold when no protein has been immobilized this is in agreement with a previous study.' The lack of any change to STM images upon adsorption of water to bare gold suggests that such features are indicative of the substrate. For a hydrophilic, insulating mica surface, Guckenberge$ and colleagues observed suprisingly high conductivity upon the adsorption of water to the bare surface containing adsorbed DNA molecules. The presence of adsorbed. possibly ordered water both on the substrate surface and adsorbed to the DNA resulted in successful imaging at very low tunneling currents (< I PA). A number of interesting findings may be drawn from the experimental data observed in Figures 3 and 4. No reversal of STM image contrast was observed upon dehydration of the sample, which is contrary to our previous studies? However in the previous study the protein layers were approximately three times thicker than those we report here, and in this case we believe the sample film thickness to be monomolecular. There is currently no unified mechanism to explain image contrast formation of proteins, and changes to image contrast can be ~ariable.2~"Therefore. it is important to bear in mind the nature of the system as well as the conditions under which the protein is being imaged. The self-assembled monolayer on gold will itself bind water, and the media from which this monolayer has been deposited may result in differing wetting characteristics, depending upon the chemical and physical properties of this
STM Images of Covalently Immobilized Proteins
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Figure 4. Plot of increase in apparent z-height (nm) as a function of the relative humidity (RH).
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significant events that are associated with regions I, 11, and 111 of the isotherm. Sorption measurements on model polymers4 and chemically modified protein^^^,^^ have suggested a molecular basis for this. The main sorption events are as follows: I, high-affinity binding of water to ionizable residues on the protein surface. In this region,
