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Characterization of a Cysteine-Containing Peptide Tether Immobilized onto a Gold Surface Tracey Baas,† Lara Gamble,‡ Kip D. Hauch,‡ David G. Castner,*,‡,§ and Tomikazu Sasaki*,† Departments of Chemistry, Bioengineering, and Chemical Engineering, University of Washington, Seattle, Washington 98195 Received December 26, 2001. In Final Form: April 11, 2002 One side of a triangular molecular tile has been successfully synthesized. The synthesized peptide contains two cysteines (Cys), two glutamic acids (Glu), and a p-aminomethylbenzoyl-p-benzoic acid (AMBBA). The cysteines anchor the peptide to a gold surface by formation of gold-thiolate bonds. The negatively charged glutamic acids should assist in the binding of positively charged proteins to the peptide. The rigid AMBBA provides a benzophenone group for photochemically tethering proteins to the peptide and also acts as the corner for the triangle. Electron spectroscopy for chemical analysis (ESCA) and near-edge X-ray absorption fine structure were used to characterize the composition and orientation of the AMBBA-CysGlu-Cys-Glu peptide on the gold surface. Adsorption from 7 µM solutions of the peptide in phosphatebuffered saline produced peptide overlayers with the thiol groups from both cysteines bound to the gold surface. The amide groups of the peptide backbone and the aromatic rings of the AMBBA groups were both oriented parallel to the gold surface. ESCA and fluorescence microscopy demonstrated that albumin could be successfully photoimmobilized to the adsorbed peptide tether. The AMBBA-Cys-Glu-Cys-Glu peptide on gold also could photochemically tether chymotrypsin. The tethered chymotrypsin was active and capable of cleaving a fluorogenic substrate. When the chymotrypsin was tethered as a positively charged protein, it exhibited higher enzyme activity than when it was tethered as a negatively charged protein. However, in all cases the activity of the bound chymotrypsin was lower than its activity in solution.
1. Introduction The controlled modification and patterning of surfaces will continue to be the basis for the advancement of protective layers, biomimetic catalysts, and nonfouling biosurfaces. While it is relatively straightforward to incorporate specific ligands into surfaces, a greater challenge is to control the density and spacing of these ligands to achieve proper cellular response. Multiple specific ligand-receptor recognition events and the relative distribution of receptor and ligand, in terms of both quantity and spatial presentation, need to be considered.1,2 We have designed a novel cyclic peptide (molecular tile [Figure 1]), to bind to surfaces and self-assemble to generate a well-defined nanoscale pattern of biorecognition groups.3 The Cys thiol groups attach the tile to the gold, inducing a β-conformation as the Cys side chains point * To whom correspondence should be addressed. David G. Castner, Department of Chemical Engineering, University of Washington, Box 351750, Seattle, WA 98195-1750. Phone: (206) 543-8094. Fax: (206) 543-3778. E-mail:
[email protected]. Tomikazu Sasaki, Department of Chemistry, University of Washington, Box 351750, Seattle, WA 98195-1700. Phone: (206) 5436590. Fax: (206) 685-8665. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Bioengineering. § Department of Chemical Engineering. (1) (a) Herbert, C. B.; McLernon, T. L.; Hypolite, C. L.; Adams, D. N.; Pikus, L.; Huang, C. C.; Fields, G. B.; Letourneau, P. C.; Distefano, M. D.; Hu, W. S. Chem. Biol. 1997, 4, 731. (b) Hypolite, C. L.; McLernon, T. L.; Adams, D. N.; Chapman, K. E.; Herbert, C. B.; Huang, C. C.; Distefano, M. D.; Hu, W. S. Bioconjugate Chem. 1997, 8, 658. (c) Massia, S. P.; Hubbell, J. A. J. Cell Biol. 1991, 114, 1089. (d) Mrksich, M. Cell. Mol. Life Sci. 1998, 54, 653. (e) Humphries, M. J. J. Cell Sci. 1990, 97, 585. (2) Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell Sci. 2000, 113, 1677. (3) (a)Boeckl, M. S.; Baas, T.; Fujita, A.; Hwang, K. O.; Bramblett, A. L.; Ratner, B. D.; Rogers, J. W.; Sasaki, T. Biopolymers 1998, 47, 185. (b) Kunitake, M.; Batina, N.; Itaya, K. Langmuir 1995, 11, 2337. Kunitake, M.; Akiba, U.; Batina, N.; Itaya, K. Langmuir 1997, 13, 1607.
down and the Xyz side chains point up. The tile’s modular design allows for a large number of variations, in both size and composition, for controlling the local surface coverage. We describe in this paper the preparation and self-assembly of the precursor of the molecular tile: the peptide tether. The precursor AMBBA-Cys-Glu-Cys-Glu is expected to attach and position the peptide horizontally to the gold by both cysteine thiols, which is crucial to the implementation of the molecular tile design. The photochemical attachment of bovine serum albumin (BSA) and chymotrypsin to the modified surface serves as a model system to demonstrate the specific protein immobilization mediated by the molecular tile. 2. Experimental Section General Methods. Samples consisting of 1000 Å of gold with no adhesion layer were prepared by sputtering gold onto plasmacleaned glass microscope slides,4a electron spectroscopy for chemical analysis (ESCA) spectra were obtained either on a Surface Science Instruments X-Probe or S-Probe spectrometer,4b and near-edge X-ray absorption fine structure (NEXAFS) experiments were performed at the National Synchrotron Light Source U7A beamline located at Brookhaven National Laboratory,5 all utilizing parameters described previously. The synthesis and characterization of AMBBA-Fmoc and Glu-Cys-Glu-CysAMBBA have been described elsewhere.2 Gold slides were placed in various dilutions of peptide in 10 mM phosphate buffer at pH 6. After 24 h, they were rinsed and held in fresh buffer until analyzed by ESCA or NEXAFS. Tethering of Albumin. For ESCA studies, gold-immobilized peptide samples received 2 mg/mL of albumin in phosphatebuffered saline (PBS). Irradiation took place for 2 h using a Spectroline 36-380 pencil lamp having an average intensity of (4) (a) Boeckl, M. S.; Bramblett, A. L.; Hauch, K. D.; Sasaki, T.; Ratner, B. D.; Rogers, J. W., Jr. Langmuir 2000, 16, 5644. (b) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 5083. (5) Nelson, K. E.; Gamble, L.; Jung, L. S.; Boeckl, M. S.; Naeemi, E.; Golledge, S. L.; Sasaki, T.; Castner, D. G.; Campbell, C. T.; Stayton, P. S. Langmuir 2000, 17, 2807.
10.1021/la011847r CCC: $22.00 © 2002 American Chemical Society Published on Web 05/15/2002
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Figure 1. A schematic showing the triangular molecular tile design with each side composed of four natural amino acids and the corner AMBBA amino acid. The structure of this molecular tile should allow hydrogen bonding between neighboring tiles, producing a nanopatterned surface. 2000 µW/cm2 at 365 nm. For fluorescent studies, the goldimmobilized peptide samples were covered with a solution of 0.1 mg/mL Oregon green-labeled albumin and heated to give a layer of dried protein. Controlled irradiation of samples was done with a Nikon Eclipse TE200 inverted or E800 upright epifluorescent microscope equipped with a 100 W mercury arc lamp. Tethering of Chymotrypsin for Enzymatic Studies. Peptide-gold samples received 2 mg/mL of chymotrypsin in a 10 mM appropriate buffer and were irradiated for 2 h with the pencil lamp. Samples were then washed repeatedly with buffer and stored in fresh buffer. For enzymatic assays, each sample was placed in 8 mL of fresh 10 mM phosphate buffer at pH 7.8, received 80 µL of a 300 µg/mL stock solution of chymotrypsin substrate II, and was held at 37 °C for 1 h. The solution was pipetted into a test tube and held at 100 °C for 5 min.
3. Results and Discussion Composition of the Immobilized AMBBA-Cys-GluCys-Glu. ESCA elemental composition scans were taken of gold “blanks” that had been immersed in DMF, ethanol, and phosphate buffer to determine the best solvent system for peptide assembly. The signal from the gold substrate is not included in the ESCA composition tables so the determined composition of the organic overlayers can be compared directly to the theoretical values expected from the stoichiometric composition of the peptide. The results in Table 1 from the blank gold surfaces indicate possible
Table 1. ESCA-Determined Atomic Percentages for Blank Gold Samples Exposed to Pure Solvents carbon oxygen nitrogen sulfur a
phosphate buffer
ethanol
DMF
theoretical
80.5 16.9 nda 2.6
74.6 20.1 2.2 3.1
79.2 13.9 3.2 3.7
63.3 22.4 10.2 4.1
nd ) not detected.
contaminants that can be deposited from the solvents and compete with peptide immobilization. The results in Table 1 show that phosphate buffer is the best choice for the peptide adsorption studies since no nitrogen signal was detected on the phosphate buffer blank, so all increases in nitrogen on a gold surface in phosphate buffer can be attributed to adsorbed peptide. For evaluation of the ESCA results from the peptide-covered surfaces, the higher the peptide surface coverage, the more closely its ESCA composition should match its theoretical composition. The results in Table 2 from the AMBBA-Cys-Glu-Cys-Glu peptide covered surfaces show that the peptide in phosphate buffer gave an elemental composition that was the closest to the theoretical composition, indicating that the peptide had immobilized to the gold surface. Therefore, the phosphate buffer was used for all subsequent peptide immobilization.
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Table 2. ESCA-Determined Atomic Percentages for Gold Samples Exposed to Solvents Containing the AMBBA-Cys-Glu-Cys-Glu Peptide carbon oxygen nitrogen sulfur
phosphate buffer
ethanol
DMF
theoretical
66.9 16.5 10.8 5.7
72.7 18.3 6.4 2.6
78.1 16.3 3 2.5
63.3 22.4 10.2 4.1
To determine what peptide concentration would give adequate surface coverage while still allowing both thiols to attach to the gold surface, samples were immersed in different concentrations of peptide in phosphate buffer. Figure 2 shows the ESCA survey spectra and highresolution S2p spectra (inset) for gold surfaces modified with two different concentrations of peptide. With ESCA, the atomic percentage of nitrogen (from the amide backbone) was used to determine if the peptide had actually bound to the gold. At high peptide concentrations (70 µM), the N1s atomic percentage of 10.8% indicates successful attachment of peptide to the gold surface, but both bound (162 eV) and unbound (164 eV) sulfur are present on the surface (see inset). This could be due to multilayer formation that hinders the thiols’ attachment
to the gold surface. As the peptide concentration is lowered (7 µM, 35 000 pmol), the unbound thiol signal becomes extremely small, indicating that both thiols are bound to the gold. The ESCA C1s and N1s atomic percentages are slightly higher and lower, respectively, than the expected theoretical percentages, indicating that some contamination is present along with the peptide on this surface. However, the strong ESCA N1s signal (7.5 atomic %) indicates that most of the gold surface is covered by peptide and not contamination. A quantity of 230 pmol of peptide (a 6 Å × 12 Å rod) is necessary to form a complete monolayer on a 1 cm2 surface. Thus, 7 µM peptide solutions were used for all further studies. Orientation of the Immobilized AMBBA-Cys-GluCys-Glu. NEXAFS experiments were done to determine the peptide orientation on the gold surface. Normal (incident X-ray beam 90° to the surface) and grazing angle (20°) NEXAFS spectra were acquired. The carbon K-edge spectra and the nitrogen K-edge spectra indicate a preferential orientation of the peptide on the surface (Figure 3). The two sharpest peaks in the carbon K-edge spectra were at ∼288 and ∼285 eV. The peak at 288 eV is assigned to the C1s to π* transition from the amide
Figure 2. ESCA survey scans of AMBBA-Cys-Glu-Cys-Glu immobilized onto gold surfaces from peptide concentrations of 70 µM (upper figure) and 7 µM (lower figure) in phosphate buffer. The insets for each concentration show the corresponding S2p highresolution spectra and overlayer composition. The gold concentration is not included so the ESCA composition can be compared directly to the theoretical values.
Cysteine-Containing Peptide Tether on Gold
Langmuir, Vol. 18, No. 12, 2002 4901 Table 3. ESCA-Determined Atomic Percentages for the Gold-Immobilized Peptide As Prepared, after Exposure to Albumin with UV Irradiation, and after Exposure to Albumin without UV Irradiationa
carbon oxygen nitrogen sulfur
goldimmobilized peptide
albumin with UV irradiation
albumin without UV irradiation
peptide theoretical
76.3 ( 1.3 14.5 ( 0.4 5.8 ( 0.3 3.5 ( 1.3
69.5 ( 0.1 15.3 ( 1.2 12.7 ( 0.1 2.2 ( 0.4
76.2 ( 2.6 13.8 ( 0.3 6.4 ( 0.8 3.1 ( 1.8
63.1 22.3 10.2 4.1
a The sample that was irradiated shows a large increase in the nitrogen concentration, consistent with the attachment of albumin.
Figure 3. NEXAFS spectra for the AMBBA-Cys-Glu-Cys-Glu peptide immobilized onto gold surfaces. The top panel shows the C K-edge spectra, and the lower panel shows the N K-edge spectra. The spectra were taken at grazing angle and normal angle to determine the orientation of the peptide. The variation in peak intensity with angle indicates that the amide bonds are parallel to the gold surface, as shown in the molecular diagram.
bonds and also includes a transition from the C-H bonds.6 The 285 eV peak is assigned to the C1s to π* transition from the aromatic rings.6 The peak at 288 eV was most intense at grazing angle, indicating that the peptide is positioned in a β form with the carbonyl groups and some of the C-H groups, parallel to the gold surface (see Figure 3), as expected. The peak at 285 eV was more intense at grazing angle, indicating that the phenyl rings of the benzophenone appeared to be positioned parallel to the gold. The nitrogen K-edge spectra confirmed the orientation of the amide orbitals. A sharp peak at ∼400 eV, attributed to the N1s to π* transition of the amide bond,6a showed more intensity at the glancing angle, consistent with the positioning of the amide bonds parallel to the gold surface. Albumin Tethering. The benzophenone moiety of the AMBBA on the gold surface can be used to attach a biomolecule.7 Albumin was used to optimize the protein attachment conditions. Albumin is present in the circula(6) (a) Urquhart, S. G.; Hitchcock, A. P.; Priester, R. D.; Rightor, E. G. J. Polym. Sci., Part B: Polym. Phys. 1995, 33, 1603. (b) Boese, J.; Osanna, A.; Jacobsen, C.; Kirz, J. J. Electron Spectrosc. Relat. Phenom. 1997, 85, 9. (c) Jordan-Sweet, J. L.; Kovac, C. A.; Goldberg, M. J.; Morar, J. F. J. Chem. Phys. 1986, 89, 2482. (d) Carravetta, V.; Oleksandr, P.; Agren, H. J. Chem. Phys. 1998, 109, 1456. (7) (a) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Cerber, Ch.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathier, H. J. Langmuir 1996, 12, 1997. (b) Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661. (c) Sigrist, H.; Collioud, A.; Clemence, J. F.; Gao, H.; Luginbuhl, R.; Sanger, M.; Sundarababu, G. Opt. Eng. 1995, 34, 2339.
tory system,8 has been used in creating stealth surfaces,9 and has been shown to interact with negative surfaces.10 Gold-immobilized peptide samples were placed in PBS with 2 mg/mL albumin and irradiated while control samples were not irradiated to ensure that the immobilized albumin was covalently bound to the surface. All samples were rinsed repeatedly with PBS after irradiation to remove any physisorbed protein. The samples that were not irradiated showed an atomic composition similar to the peptide-gold surfaces, while the irradiated samples had a significant increase in nitrogen content (Table 3). To visualize the protein tethering, the gold-immobilized peptide samples were covered with Oregon green-labeled albumin and heated to produce a layer of dried protein. These samples were then irradiated at wavelengths less than 515 nm and washed repeatedly with 1% sodium dodecyl sulfate (SDS) to remove any physisorbed protein. Circles of immobilized protein, corresponding to various beam sizes, were observed (Figure 4). The samples that had been irradiated for 20 min showed a darker circle in the middle of the immobilized fluorescent protein due to photobleaching. The fluorophore is not bleached on the outer ring due to the lower intensity of the focused beam in that region. The sample that had been irradiated for 10 min using the field stop showed less photobleaching and a smaller circle corresponding to the smaller size of the beam emitted through the mask of the field stop. These data show that the peptide on the gold slide can immobilize the fluorescently labeled protein via a covalent tether upon photoactivation. Activity of Tethered Chymotrypsin. We used SucAla-Ala-Pro-Phe-AMC, chymotrypsin substrate II,11 as our substrate to detect chymotrypsin activity on the surface. The peptide substrate shows an emission peak at 385 nm. When the peptide is hydrolyzed and the AMC is free, the emission peak changes to 445 nm. Samples of immobilized peptide on gold were placed in a solution of chymotrypsin in phosphate buffer, pH 7. The enzyme (pI ) 8.75) should be positively charged and attracted to the peptide.12 The gold control samples exposed to chymotrypsin without irradiation showed no enzymatic activity; all emission was at 385 nm (Figure 5). The gold-immobilized peptide sample immersed in chymotrypsin that had been irradiated showed fluorescent emission at 445 nm (cleaved substrate) and at 385 nm (unreacted substrate). The slide retained enzymatic activity for a series of assays, indicating that the chymotrypsin was covalently attached to the surface. These (8) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153. (9) Engbers, G. H.; Feijen, J. Int. J. Artif. Organs 1991, 14, 199. (10) Blomberg, E.; Claesson, P. M.; Froberg, J. C. Biomaterials 1998, 19, 371. (11) (a) Turner, D. C.; Testoff, M. A.; Conrad, D. W.; Gaber, B. P. Langmuir 1997, 13, 4855. (b) Irvine, G. B.; Ennis, M.; Williams, C. H. Anal. Biochem. 1990, 185, 304. (12) Ui, N. Biochim. Biophys. Acta 1971, 229, 582.
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Figure 5. Typical fluorescence spectra used to determine the enzymatic activity of different surfaces for the chymotrypsin substrate II. The gold control sample and gold-immobilized AMBBA-Cys-Glu-Cys-Glu peptide sample immersed in chymotrypsin without UV irradiation both have peaks in the fluorescence spectra at 385 nm due to unreacted substrate. The gold-immobilized peptide sample immersed in chymotrypsin with UV irradiation shows a new emission peak at 445 nm due to cleaved substrate. The peak at 385 nm in this spectrum is due to unreacted substrate.
had been formed, 800 ng would be available for reaction. The small amount of observed active protein could be due to submonolayer coverage of protein, obstruction of the active site upon binding, or an inactivation of protein upon tethering. 4. Conclusion
Figure 4. Two gold samples with immobilized AMBBA-CysGlu-Cys-Glu peptides that have been irradiated to tether fluorescently labeled albumin. The dark circle in the center is due to photobleaching. The small circle in the lower left-hand corner of the top photo was created using a field stop.
results show that the peptide tether has the ability to create bioactive surfaces that are reusable. Next, the same immobilization scheme was tried in Trizma buffer at pH 7, where the enzyme should be positively charged, and Trizma buffer at pH 10, where the enzyme should be negatively charged. The samples that were irradiated in the pH 7 buffer showed more activity than those irradiated in the pH 10 buffer. In a comparison of the amount of substrate cleaved by immobilized chymotrypsin to that cleaved by chymotrypsin in solution, the amount of active enzyme on the surface was determined to be between 100 pg and 10 ng. If a full monolayer of chymotrypsin (50 Å × 40 Å × 40 Å protein13) (13) Appel, W. Chymotrypsin: Molecular and Catalytic Properties; Clinical Biochemistry Vol. 19; 1986; pp 317-323.
The peptide AMBBA-Cys-Glu-Cys-Glu, one side of a triangular molecular tile, was successfully synthesized. ESCA and NEXAFS experiments demonstrated that this peptide adsorbs to the gold surface via two thiolate bonds with the peptide’s amide backbone parallel to the gold surface. Successful photochemical immobilization of albumin to the adsorbed peptide tether was demonstrated by ESCA and fluorescence microscopy experiments. The adsorbed AMBBA-Cys-Glu-Cys-Glu peptide was also shown to photochemically tether chymotrypsin with some retention of its enzyme activity, as indicated by the cleavage of a fluorogenic chymotrypsin substrate. When the chymotrypsin was tethered as a positively charged protein, it had higher enzyme activity than when it was tethered as a negatively charged protein, suggesting that the peptide sequence and charge can be used to attract proteins to the benzophenone tethering site. While this study has examined just one side of the molecular tile, it is anticipated that the full molecular tile would bind to the gold surface in a similar manner. Acknowledgment. This work was supported by the University of Washington Engineered Biomaterials (UWEB, NSF Grant EEC-9529161) and the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO, NIH Grant RR01296). NEXAFS studies were performed at the NSLS, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Science and Division of Chemical Sciences. The expertise of Deborah LeachScampavia and Ariana Bramblett (ESCA) is gratefully acknowledged. Helpful discussions with Anthony Sartori and Dr. Maximiliane Boeckl are respectfully acknowledged. LA011847R
