Langmuir 1994,10,2860-2862
2860
Site-SpecificImmobilization of Molecularly Engineered Dihydrofolate Reductase to Gold Surfaces Stephen J. Vigmond,t Masahiro Iwakura,J:Fumio Mizutani,' and Tatsuo Katsura*>? National Institute of Bioscience a n d Human-Technology a n d National Institute of Advanced Interdisciplinary Research, 1-1Higashi, Tsukuba, Ibaraki, J a p a n 305 Received February 7,1994. I n Final Form: May 31, 1994@ The introduction of accessible cysteine residues by genetic engineering techniques is demonstrated to be a viable method to immobilize proteins onto gold surfaces in a controlled manner. DHFR-AS, a dihydrofolate reductase mutant which has had both native cysteine residues replaced, exhibited only limited adsorption as determined by thickness-shear mode piezoelectric sensor and enzymatic assay measurements. With the addition of a cysteine residue to the C-terminal end of the enzyme (DHFRAS-C), the level of adsorption was increased by a factor of approximately 4. The coverage of DHFR-AS-C mol/cm2. over the gold surface is estimated to be 4 x
Introduction The immobilization of proteins onto a variety of surfaces is a n active area of research that is useful for a range of applications. A general methodology that is currently being utilized for this purpose is the self-assembly of various functional groups onto specific surfaces such as the reaction of thiols with gold. This type of interaction allows one to design immobilization schemes through either weak adsorptionlJ or the formation of covalent bond^.^-^ However, a limitation of these methods is their nonspecificity; the nature of the noncovalent adsorptions is poorly defined and probably incorporates many orientations while covalent attachment typically occurs through one of several reactive groups (such as a n amino functionality) on the surface of the protein so there will still be a set of possible orientations. The recent availability of genetic engineering techniques provides a n opportunity to introduce single immobilization sites onto protein surfaces which will allow interfaces to be formed with much greater regularity. Sliger and coworkers have produced two cytochrome bg mutants by site-directed mutagenesis that have positioned cysteine groups on opposite sides of the protein surface. Differential heme orientation, with respect to a Ti:Zn glass substrate which had been derivatized with a silane coupling agent, was confirmed by the two assemblies generating linear dichroism values with opposite relative signs.6 The same mutants were also immobilized on a n agrose chromatography support and shown to be capable of imparting some control over the macromolecular recognition between the cytochrome bg and cytochrome c.' Previously we have reported that the introduction of a n extra cysteine residue onto the C-terminal end of dihyrofolate reductase (DHFR) increased the reactivity of the thiol group and thereby increased the amount of t National Institute of Bioscience and
Human-Technology.
National Institute of Advanced Interdisciplinary Research. Abstract published inAdvance ACSAbstracts, August 15,1994. (1) Salamon, Z.;Hazzard, J. T.; Tollin, G. Proc. NatLAcad.Sci. U S A . @
1993, 90, 6420. (2) Kinnear, K. T.; Monbouquette, H. G. Langmuir 1993, 9, 2255. (3) Song, S.;Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J . Phys. Chem. 1993, 97, 6564. (4) Hyndman, D.; Lever, G.; Burrell, R.; Flynn, T. G. Biotechnol. Bioeng. 1992, 40, 1319. (5)Leggett, G. J.; Roberts, C. J.; Williams, P. M.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. Langmuir 1993, 9, 2356. ( 6 ) Stayton, P. S.; Olinger, J. M.; Jiang, M.; Bohn, P. W.; Sliger, S. G . J . Am. Chem. SOC.1992,114, 9298. (7) McLean, M. A.; Stayton, P. S.; Sliger, S. G.Ana1.Chem. 1993,65, 2676.
0743-7463/94/2410-2860$04.50/0
enzyme which was effectively immobilized by a thiopropylSepharose I n this paper, we use engineered DHFR to demonstrate that this scheme can be extended to metallic substrates; the accessible cysteine residues can bind to gold surfaces through the reaction of the thiol moiety. We compare the adsorption of a n engineered DHFR species (DHFR-AS) which has had both natural cysteine residues replaced (Cys85 to Ala and Cys152 to Ser) with t h a t of a simular mutant (DHFR-AS-C) which has had a cysteine residue added directly to the terminus (Cysl60) (Figure 1). From frequency changes obtained by adsorbing the proteins onto thickness-shear mode (TSM) piezoelectric sensors and the corresponding enzyme assay measurements, the presence of the cysteine group is seen to enhance the level of immobilization.
Experimental Section DHFR-ASand DKFR-AS-C ( M W approximately 18 000)were prepared similarlyto that described previously and the primary structures were confirmedby nucleotide sequencing.9J0 In order to obtain freshly-reduced samples, 5 mL of the enzyme mixture are centrifuged at 15 000 rpm for 20 min and then the supernatant is decanted off. The residue is dissolved in 1.0 mL of degassed 25 mM EDTA pH 8 solution before adding 10 pL of 0.1 M dithiothreitol. After the mixture has been at room temperature for at least 2 h, the dithiothreitol is removed by gel filtration chromatography using a Sephadex G50 column and deoxygenated 25 mM EDTApH 8 as the eluent. The fractions are analyzed for DHFR by U V spectroscopy using a wavelength of 280 nm = 31 The 5-MHz gold-coated TSM sensors (Hokuto-Denko Co., Tokyo)have a total surface area of 2.30 cm2(ignoringroughness factors),ofwhich 1.05cm2 is gold and 1.25cm2is exposed quartz. They are cleaned by rinsingin acetone and then soaking in ethanol until stable frequency readings are obtained. Any remaining adventitious surface hydrocarbons will be removed by the chemisorption process.12 After the TSM sensors are immersed in the enzyme solutionsat 4 "C for various times, they are rinsed with distilled water, soaked in 1M KC1 for 20 min, and washed 3 times in water and finally 3 times in ethanol before drying by standing in air. Short-term exposures to ethanol do not affect the enzymes. The frequencies and enzyme activities of the sensors are then measured. The solution used for the enzyme (8) Iwakura, M.; Kokubu, T. J.Biochem. 1993,114, 339. (9) Iwakura, M.; Furusawa, K.; Kokubu, T.; Ohashi, S.; Tanaka, Y.; Shimura, Y.; Tsuda, K. J . Biochem. 1992,111,37. (10) Iwakura, M.; Jones, B.; Luo,J.;Matthews, C. R. Manuscript in preparation. (11) Touchette, N. A.; P e w , K. M.; Matthews, C. R. Biochemistry 1986,25,5445. (12) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.;Whitesides, G . M.; Nuzzo, R. G. J . Am. Chem. SOC.1989,111, 321.
0 1994 American Chemical Society
Langmuir, Vol. 10,No. 9, 1994 2861
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Figure 1. Schematic representations of DHFR-AS (top) and
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DHFR-AS-C (bottom). The shaded boxes indicate the relative positions of the two native cysteine residues whichwere replaced with Ala and Ser. assay13is prepared by dissolving 1.0 mg of dihydrofolic acid, 4.0 mg of NADPH and 50 pL of 2-mercaptoethanol in 50 mL of 25 mM phosphate pH 7 buffer that has been deoxygenated by aspiration. After cooling a 3.0-mL aliquot of this assay solution to 15 “C,a coated-TSM sensor is dropped into the solution. A reaction time between 30 and 60 min is employed (an estimate is made to keep the measured absorbance decrease below 0.11, at which time the reaction is severely retarded by cooling in an ice bath. The enzyme activity is determined by the absorbance decrease measured at 340 nm. For the purpose of control experiments, quartz substrates are obtained by using emery paper to remove the gold electrodes from the TSM sensors and treated in the same manner as the gold-coated devices.
Results and Discussion The ability of the cysteine residue to increase the level of attachment of active enzyme to the gold surface is obvious from a comparison of DHFR-AS-C with the cysteine-less analog DHFR-AS (Figure 2). Soaking in DHFR-AS solution produces only limited changes in both the frequency and enzymeassay measurements with time; with no cysteine group present, there is a maximum frequency decrease of about 40 Hz and a maximum absorbance change at 340 nm of approximately 0.04 AU/ h. On the other hand, DHFR-AS-C can cause frequency changes of approximately200 Hz and absorbance changes as high as 0.2 AUh. Within 30 s of submersion, both enzymes cause a frequency decrease of approximately 30 Hz and an absorbance decrease of approximately 0.1AUh which suggests that both enzymes initially adsorb nonspecificallyto the gold surface. With no accessiblecysteine group though, “permanent”attachment to the surface will require many nonspecific, weak attachment sites and hence limits to the amount of enzyme that may be bound. With the covalent sulhr-metal bond, fewer attachment sites will be necessary and thereby permit more efficient packing of the enzyme so more enzyme will be able to bind to the surface. Both the TSM and enzyme assay results indicate that even &er 200 h there are still some changes occurring in the adsorbed film. The relatively large size of the enzymes require very long times to optimize their packing on the surface. As the procedure used here requires the gold surfaces to be removed from the enzyme solution and washed before the frequency and absorbance measurements are made, there is obviously some retardation of the enzyme adsorption. Upon reimmersion into the enzyme solution, a certain amount of time is required to reestablish the surface to the state it was in before removing it from the solution. For this reason, after (13)Hillcoat,B. L.; Nixon, P. F.; Blakley, R. L. Anal. Biochen. 1976, 21, 178.
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Figure 2. Frequency changes of TSM sensors (A) and the corresponding absorbance changes of enzymatic assay mixtures at 340 nm (B) as a function of immersion time in enzyme solutions. DHFR-AS data (+) are a combination of three trials using solutions at concentrations between 1.7 and 2.1 mg/mL while DHFR-AS-C data (0)are a combination of four trials using solutions at concentrations between 1.5 and 2.1 mg/mL. The absorbancechanges (0)solelydue to DHFR-AS-C adsorbed
onto the quartz part of TSM sensor are also plotted.
determining some of the effects on the gold surfaces from short-term immersions in the enzyme solutions, measurementswere limited to about every second day. Under these conditions, the adsorption requires on the order of 200 to 300 h. As the frequency changes of TSM sensors are a function of both mass changes and viscoelastic properties,14estimations of the amount of immobilized enzyme were made from the enzyme assay measurements. While the area responsiblefor the frequencyresponse of the TSM devices is restricted to the area where the gold electrodes overlap (the sensitivity of the sensor decreases exponentiallyfrom the center), all adsorbed enzyme onto both the gold electrodes and the exposed quartz surface will contribute to the measured enzymatic activity so the enzyme-quartz interaction must also be considered. The quartz substrates obtained by removing the gold electrodes from the TSM sensors were used to measure the enzyme-quartz interaction. The absorbance changes due to the enzyme adsorbed onto the quartz part of the TSM sensor were estimated from the known areas of each parts (roughness factorswere not taken into consideration)and are included in Figure 2B. Although the amount of adsorption onto the quartz surface is greater with the enzyme containing the accessible cysteine residue (data not shown), the contributionfrom the enzyme adsorbed on the quartz part (14) Grate, J.W.; Klusty, M.; McGill, R. A.; Abraham, M. H.; Whiting, G.; Andonian-Haftvan, J. Anal. Chem. 1992,64,610.
2862 Langmuir, Vol. 10, No. 9, 1994
Letters
of the TSM sensor is quite less than that from the enzyme on the gold electrode surface. Recent work has shown the possibility of chemisorption of thiol derivatives onto oxide substrates.15 Enzyme assays measurements were carried out with the solutions of the engineered enzymes under the same conditions used to monitor surface adsorption. Using both ofthe enzymes, solution activities are 0.06 f0.02 DAUh for 40-ng enzyme trials (standard deviation from 6 trials) and 0.18 f 0.04 DAUh for 100-ng enzyme trials (5 trials). This indicates that nonspecific adsorption of DHFR-AS binds approximately 25 ng of enzyme to the TSM surface but DHFR-AS-C, with a n accessible cysteine group, a t concentrations of approximately 2 mg/mL, can achieve levels greater than 100 ng over both sides of the piezoelectric crystal. These masses correspond to 1 x and 4 x mol/cm2 over the gold areas, respectively. Further, the higher density coverage correlates to a n average area of 4 x lo3 A2/moleculewhich is reasonable for DHFR since the molecule may to a first-approximation, be represented as a sphere of 50 diameter, judging from the X-ray structure.16 It was observed that the 2-mercaptoethanol included in the assay solution dissolves some of the,gold from the TSM sensors which increases the frequency one measures. A 14 mM solution of2-mercaptoethanol in 0.1 M phosphate pH 7 (equivalent to the enzyme assay solution) a t 4 "C will cause a frequency increase of approximately 100 Hz over 24 h; the effect is increased a t higher temperatures but reduced if the surface is coated with enzyme. The removal of the gold was confirmed by atomic absorption spectrometry and this result agrees with other reports that thiol compounds can dissolve g01d.17J8 However, if the enzymatic assay reactions are kept to a minimum (1
h for the initial measurement and then reduced to 30 min when the activity becomes greater), no significant frequency increases are detectable even with uncoated TSM devices that are used as references. Although the previous work showed another DHFR derivative to be able to retain its activity as a dried gel (Sepharose 6B) for 3 years, the enzyme coatings formed here do not exhibit such a supreme stability. Storage in 1M KC1, ethanol, or the 25 mM EDTA pH 8 buffer all lead to lower measured activities although the frequency readings remain essentially the same. This combination of facts suggests that the enzyme remains immobilized so the lower activity must result from some inactivation process on the gold surface. It also appears that the inactivation does not involve any gross structural changes, as this would induce significant frequency changes. Both the TSM sensor and enzymatic assay measurements indicate much greater adsorption with DHFRAS-C than DHFR-AS which confirms the ability of the cysteine residue on the enzyme surface to bind to gold surfaces. Presently, we are further pursuing these immobilizations using DHFR mutants with different spacers to determine iflonger chains will allow the enzyme to reorient more easily and improve the packing density or change the rate of adsorption. Modifications of the metallic surface are also being performed in a n attempt to improve the stability of the immobilized enzyme. This type of experiment should have direct relevance to general studies of protein adsorption to surfaces. The use of enzymes, which will be subject to a high degree of intermolecular interactions during adsorption to the surface, will slow the kinetics of the chemisorption and may provide insight into the mechanism of the selfassembled monolayer formation of smaller molecules.
(15)Sato, Y.; Uosaki, I€ Denki Kagaku 1993,61, 816. (16)Bystroff, C.; Oatley, S. J.;Kraut, J.Biochemistry 1990,29,3263. (17)McCarley, R. L.;Kim, Y.-T.; Bard, A. J. J. Phys. Chem. 1993, 97,211. (18)Edinger, K.; Golzhauser, A.; Demota, K.; Wo11, Ch.; Grunze, M. Langmuir 1993,9,4.
Acknowledgment. The authors thank Dr. Soichi Yabuki of the National Institute of Bioscience and HumanTechnology for his technical assistance and helpful discussions.
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