Langmuir 2008, 24, 5795-5801
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Structural Information of Mussel Adhesive Protein Mefp-3 Acquired at Various Polymer/Mefp-3 Solution Interfaces Mark A. Even, Jie Wang, and Zhan Chen* Department of Chemistry, 930 North UniVersity AVenue, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed January 15, 2008. ReVised Manuscript ReceiVed March 9, 2008 Mytilus edulis foot protein Mefp-3 serves as a primer in the formation of adhesive plaques that attach the mussel to solid surfaces in its immediate environment. The adsorption behavior of this protein on various materials of different hydrophobicity was studied using sum frequency generation (SFG) vibrational spectroscopy. By collecting SFG signals from side chains of these amino acids and from secondary structures of the protein, we have determined that this protein adopts different conformations at different interfaces, depending on hydrophobicity of the contact medium and specific chemical group interactions. We have also demonstrated that SFG has the potential to track the interfacial conformations of a single amino acid in a protein.
1. Introduction Understanding of molecular interactions at interfaces is important in fundamental research as well as in many applications such as biomaterials and biofouling. The problem of marine biofouling dates back to the earliest use of marine vessels. Marine organisms such as mussels, barnacles, and tubeworms can attach themselves to and grow on any submerged solid surface. Such fouling of ship hulls and marine equipment amounts to a widespread problem, as the resulting increase in surface roughness causes a corresponding increase in hydrodynamic drag that a vessel would experience when moving through the water. Marine biofouling costs the international community billions of dollars annually in the form of increased fuel expenses to compensate for the increased drag and substantial expenditures for hull cleaning, painting, and maintenance. Preventive measures such as copper plating and, more recently, organotin-based coatings on hulls have reduced these costs at the expense of releasing compounds into the environment that are known to be harmful to marine life.1,2 One approach that may circumvent these problems is to use polymer coatings that are specifically designed to minimize bioadhesion or to facilitate the removal of the material (biofouling release). In support of this approach, it is necessary to elucidate the mechanisms of the chemical and physical interactions between the bioadhesive and the prospective antifouling polymer. In many cases, such as with marine mussels, the bioadhesive takes the form of an adsorbed protein film. Because it is important in so many fields, protein adsorption at synthetic surfaces has been studied by many research groups for several decades using a wide variety of techniques. Work on mussel adhesive proteins is much more recent. For example, the family of proteins that comprise the adhesive plaque of the edible blue mussel Mytilus edulis have been isolated and characterized in only the past two decades, and this plaque is understood in greater detail than any other marine bioadhesive.3 The effective* To whom all correspondence should be addressed. E-mail: zhanc@ umich.edu. Fax: 734-647-4865. (1) Wynne, K. J.; Guard, H. NaV. Res. ReV. 1997, 49, 2–3. (2) Clare, A. S.; Evans, L. V. Biofouling 2000, 16, 81–82. (3) Wiegemann, M. Aquat. Sci. 2005, 67, 166–176. (4) Tatehata, H.; Mochizuki, A.; Ohkawa, K.; Yamada, M.; Yamamoto, H. J. Adhes. Sci. Technol. 2001, 15, 1003–1013. (5) Waite, J. H.; Andersen, N. H.; Jewhurst, S.; Sun, C. J. Adhes. 2005, 81, 297–317.
ness of these proteins as a form of “underwater glue” has led to investigations into their potential as a tissue adhesive.4,5 1.1. Mytilus edulis. Mytilus edulis adapts to a high energy marine environment by attaching itself to hard surfaces via byssus threads, which run from the base of the mussel foot to small adhesive pads made of the plaque proteins.6–8 A series of glands and tissues are located within the length of the foot according to the particular protein that they secrete to form the respective components of the byssus thread and adhesive plaque. Mefp-1 forms a heavily cross-linked varnish or cuticle that covers the thread and adhesive pad. Mefp-2 and Mefp-4 comprise the rubbery foam interior, which is the bulk of the proteinaceous material of the adhesive pads. Mefp-3 and Mefp-5 are the primer proteins, which govern the adhesion between the substrate and the bulk of the adhesive pad. All these proteins are related in that they contain post-translational modifications of certain amino acids, such as 4-hydroxyproline, 3,4-dihydroxyphenylalanine (Dopa), and 4-hydroxyarginine. In this group of proteins, Dopa is particularly important, as it performs two important roles in the formation and adhesive properties of the plaque. Upon secretion from the low pH interior of the mussel foot, the catechol side chain of the Dopa is oxidized at seawater pH to react with other catechols to strongly cross-link all the plaque proteins together. Dopa groups are also capable of hydrogen bonding with hydrophilic polymers and forming strong complexes with metals and minerals.9 Subsequent studies have been performed on the adsorption behavior of mussel adhesives or adhesive plaque proteins such (6) Waite, J. H. J. Adhes. Soc. Jpn. 1997, 33, 186–193. (7) Waite, J. H. Ann. N.Y. Acad. Sci. 1999, 875, 301–309. (8) Waite, J. H. Integr. Comp. Biol. 2002, 42, 1172–1180. (9) Lee, H.; Scherer, N. F.; Messersmith, P. B. Proc. Nat. Acad. Sci. U.S.A. 2006, 103, 12999–13003. (10) Hansen, D. C.; Luther, G. W.; Waite, J. H. J. Colloid Interface Sci. 1994, 168, 206–216. (11) Schnurrer, J.; Lehr, C.-M. Int. J. Pharm. 1996, 141, 251–256. (12) Hansen, D. C.; Corcoran, S. G.; Waite, J. H. Langmuir 1998, 14, 1139– 1147. (13) Ooka, A. A.; Garrel, R. L. Biopolymers 1999, 57, 92–102. (14) Suci, P. A.; Geesey, G. G. J. Colloid Interface Sci. 2000, 230, 340–348. (15) Suci, P. A.; Geesey, G. G. Langmuir 2001, 17, 2838–2540. (16) Haemers, S.; van der Leeden, M. C.; Nijman, E. J.; Frens, G. Colloids Surf., A 2001, 190, 193–203. (17) Fant, C.; Sott, K.; Elwing, H.; Hook, F. Biofouling 2000, 16, 119–132. (18) Berglin, M.; Hedlund, J.; Fant, C.; Elwing, H. J. Adhes. 2005, 81, 805– 822. (19) Gao, Z.; Bremer, P. J.; Barker, M. E.; Tan, E. W.; McQuillan, A. J. Appl. Spectrosc. 2007, 61, 55–59.
10.1021/la800138x CCC: $40.75 2008 American Chemical Society Published on Web 05/07/2008
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as Mefp-1 and Mefp-2 at hydrophilic surfaces10–19 or hydrophobic surfaces.20–26 In either case, the main spectroscopic techniques were surface-enhanced Raman spectroscopy (SERS), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, surface splasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and ellipsometry. These surface-sensitive methods have provided some interesting and useful information about the behavior of proteins at various interfaces. As mentioned, Mefp-3 and Mefp-5 are primer proteins, and in order to understand bioadhesion between Mytilus edulis and various surfaces it is necessary to investigate Mefp-3 and Mefp-5 structures at interfaces. In this work, we will apply a nonlinear optical laser technique, sum frequency generation (SFG) vibrational spectroscopy, to study structural information of Mefp-3 at solid/ liquid interfaces in situ. 1.2. SFG. Recently, SFG has been developed into a powerful tool to study interfacial structures,27–45 including proteins and peptides adsorbed onto various surfaces.46–76 Under the electricdipole approximation, SFG signals are generated in materials where inversion symmetry is absent within the optical field. The selection rules of the technique allow specific molecular level structural and orientational information to be acquired without bulk solution signal contributions or the need to subtract a water background. Recent SFG studies have established the technique as suitable for in situ protein adsorption studies,46–76 demonstrating the potential of the technique to elucidate detailed protein (20) Olivieri, M. P.; Rittle, K. H.; Tweeden, K. S.; Loomis, R. E. Biomaterials 1992, 13, 201–208. (21) Yamamoto, H.; Ogawa, T.; Ohkawa, K. J. Colloid Interface Sci. 1995, 176, 111–116. (22) Baty, A. M.; Suci, P. A.; Tyler, B. J.; Geesey, G. G. J. Colloid Interface Sci. 1995, 177, 307–315. (23) Baty, A. M.; Leavitt, P. K.; Siedlecki, C. A.; Tyler, B. A.; Suci, P. A.; Marchant, R. E.; Geesey, G. G. Langmuir 1997, 13, 5702–5710. (24) Harder, P.; Grunze, M.; Waite, J. H. J. Adhes. 2000, 73, 161–173. (25) Frank, B. P.; Belfort, G. Langmuir 2001, 17, 1905–1912. (26) Suci, P. A.; Geesey, G. G. Colloids Surf., B 2001, 22, 159–168. (27) Shen, Y. R. The Principles of Nonlinear Optics; Wiley: New York, 1984. (28) Zhuang, X.; Miranda, P. B.; Kim, D.; Shen, Y. R. Phys. ReV. B 1999, 59, 12632–12640. (29) Bain, C. D. J. Chem. Soc., Faraday Trans. 1995, 91, 1281–1296. (30) Eisenthal, K. B. Chem. ReV. 1996, 96, 1343–1360. (31) Scatena, L. F.; Brown, M. G.; Richmond, G. L. Science 2001, 292, 908– 912. (32) Gautam, K. S.; Schwab, A. D.; Dhinojwala, A.; Zhang, D.; Dougai, S. M.; Yeganeh, M. S. Phys. ReV. Lett. 2000, 85, 3854–3857. (33) Chen, Z.; Shen, Y. R.; Somorjai, G. A. Annu. ReV. Phys. Chem. 2002, 53, 437–465. (34) Wang, J.; Chen, C. Y.; Buck, S. M.; Chen, Z. J. Phys. Chem. B 2001, 105, 12118–12125. (35) Wang, J.; Paszti, Z.; Even, E. A.; Chen, Z. J. Am. Chem. Soc. 2002, 124, 7016–7023. (36) Briggman, K. A.; Stephenson, J. C.; Wallace, W. E.; Richter, L. J. J. Phys. Chem. B 2001, 105, 2785–2791. (37) Bordenyuk, A. N.; Jayathilake, H.; Benderskii, A. V. J. Phys. Chem. B 2005, 109, 15941–15949. (38) Ma, G.; Liu, D. F.; Allen, H. C. Langmuir 2004, 20, 11620–11629. (39) Fitchett, B. A.; Conboy, J. C. J. Phys. Chem. B 2004, 108, 20255–20262. (40) Ye, S.; Morita, S.; Li, G. F.; Noda, H.; Tanaka, M.; Uosaki, K.; Osawa, M. Macromolecules 2003, 36, 5694–5703. (41) Kweskin, S. J.; Komvopoulos, K.; Somorjai, G. A. Langmuir 2005, 21, 3647–3652. (42) Chou, K. C.; Kim, J.; Baldelli, S.; Somorjai, G. A. J. Electroanal. Chem. 2003, 554, 253–263. (43) Rivera-Rubero, S.; Baldelli, S. J. Phys. Chem. B 2004, 108, 15133– 15140. (44) Ye, H.; Gu, Z.; Gracias, D. H. Langmuir 2006, 22, 1863–1868. (45) Casford, M. T. L. P. B.; Davies, P. B. Langmuir 2003, 19, 7386–7391. (46) Chen, Z.; Ward, R.; Tian, Y.; Malizia, F.; Gracias, D. H.; Shen, Y. R.; Somorjai, G. A. J. Biomed. Mater. Res. 2002, 62, 254–264. (47) Kim, J.; Somorjai, G. A. J. Am. Chem. Soc. 2003, 125, 3150–3158. (48) Koffas, T. S.; Kim, J.; Lawrence, C. C.; Somorjai, G. A. Langmuir 2003, 19, 3563–3566. (49) Kim, J.; Cremer, P. S. ChemPhysChem 2001, 8/9, 543–546. (50) Kim, G.; Gurau, M.; Kim, J.; Cremer, P. S. Langmuir 2002, 18, 2807– 2811. (51) Kim, G.; Gurau, M. C.; Lim, S. M.; Cremer, P. S. J. Phys. Chem. B 2003, 107, 1403–1409.
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structures. More details regarding this technique can be found in our previous publications. 1.3. Mefp-3 Details. Mefp-3 is a family of proteins with some unusual characteristics. It is relatively small (∼6 kDa) with an isoelectric point above 10.5, and it exists in the form of at least nine variants.77Although details of the secondary structure have not yet been studied, the complete primary structure of variant F has been determined as follows: ADYYGPNYGPPRRYGGGNYNRYNRYGRRYGGYKGWNNGWNRGRRGKYW. This protein is related to the other plaque proteins in that it is very hydrophilic, but it contains even more Dopa than Mefp-1, Mefp-2, or Mefp-4. It also contains a large amount of 4-hydroxyarginine. The large number of glycines allows this small protein a high degree of conformational freedom, making the hydroxyarginines available for hydrogen bonding and the Dopas available for cross-linking reactions or contact and adhesion to the substrate.78–81 Some details of adhesion mechanics have been proposed,82 with the Dopa groups involved in hydrogen bonding, metal complexation, and π-π and π-cation interactions and the hydroxyarginine capable of forming several H-bonds as well as π-π and Coulombic interactions.
2. Experimental Section 2.1. Sample Preparation: Protein Extraction. Mussel feet frozen at -80 °C were ordered from a restaurant supplier (Northeast Transport). The feet were allowed to warm up to be transferred to cold glass plates, where they were arrayed in a manner convenient for individual dissection and then refrozen for later use. When they were dissected, the feet were allowed to warm up sufficiently to allow the epithelial layer to be scraped off and the phenol glands to be cut out and refrozen. Following a previously published (52) Jung, S. Y.; Lim, S. M.; Albertorio, F.; Kim, G.; Gurau, M. C.; Yang, R. D.; Holden, M. A.; Cremer, P. S. J. Am. Chem. Soc. 2003, 125, 12782–12786. (53) Wang, J.; Buck, S. M.; Even, M. A.; Chen, Z. J. Am. Chem. Soc. 2002, 124, 13302–13305. (54) Wang, J.; Buck, S. M.; Chen, Z. J. Phys. Chem. B 2002, 106, 11666– 11672. (55) Wang, J.; Buck, S. M.; Chen, Z. Analyst 2003, 128, 773–778. (56) Wang, J.; Clarke, M. L.; Zhang, Y. B.; Chen, X. Y.; Chen, Z. Langmuir 2003, 19, 7862–7866. (57) Clarke, M. L.; Wang, J.; Chen, Z. Anal. Chem. 2003, 75, 3275–3280. (58) Wang, J.; Even, M. A.; Chen, X. Y.; Schmaier, A. H.; Waite, J. H.; Chen, Z. J. Am. Chem. Soc. 2003, 125, 9914–9915. (59) Wang, J.; Paszti, Z.; Even, M. A.; Chen, Z. J. Phys. Chem. B 2004, 108, 3625–3632. (60) Paszti, Z.; Wang, J.; Clarke, M. L.; Chen, Z. J. Phys. Chem. B 2004, 108, 7779–7787. (61) Wang, J.; Clarke, M. L.; Chen, Z. Anal. Chem. 2004, 76, 2159–2167. (62) Dreesen, L.; Humbert, C.; Startenaer, Y.; Caudano, Y.; Volcke, C.; Mani, A. A.; Peremans, A.; Thiry, P. A. Langmuir 2004, 20, 7201–7207. (63) Dreesen, L.; Sartenaer, Y.; Humbert, C.; Mani, A. A.; Lemaire, J. J.; Methivier, C.; Pradier, C. M.; Thiry, P. A.; Peremans, A. Thin Solid Films 2004, 464-465, 373–378. (64) Clarke, M. L.; Wang, J.; Chen, Z. J. Phys. Chem. B 2005, 109, 22027– 22035. (65) Wang, J.; Chen, X.; Clarke, M. L.; Chen, Z. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4978–4983. (66) Wang, J.; Clarke, M. L.; Chen, X.; Even, M. A.; Johnson, W. C.; Chen, Z. Surf. Sci. 2005, 587, 1–11. (67) Chen, X.; Wang, J.; Sniadecki, J. J.; Even, M. A.; Chen, Z. Langmuir 2005, 21, 2262–2264. (68) Chen, X.; Clarke, M. L.; Wang, J.; Chen, Z. Int. J. Mod. Phys. B 2005, 19, 691–713. (69) Chen, X.; Chen, Z. Biochim. Biophys. Acta 2006, 1758, 1257–1273. (70) Lee, S.; Wang, J.; Krimm, S.; Chen, Z. J. Phys. Chem. A 2006, 110, 7035–7044. (71) Wang, J.; Chen, X.; Clarke, M. L.; Chen, Z. J. Phys. Chem. B 2006, 110, 5017–5024. (72) Doyle, A. W.; Fick, J.; Himmelhaus, M.; Eck, W.; Graziani, I.; Prudovsky, I.; Grunze, M.; Maciag, T.; Neivandt, D. J. Langmuir 2004, 20, 8961–8965. (73) Knoesen, A.; Pakalnis, S.; Wang, M.; Wise, W. D.; Lee, N.; Frank, C. W. IEEE J. Sel. Top. Quantum Electron. 2004, 10, 1154–1163. (74) Mermut, O.; Phillips, D. C.; York, R. L.; McCrea, K. R.; Ward, R. S.; Somorjai, G. A. J. Am. Chem. Soc. 2006, 128, 3598–3607. (75) Chen, X.; Wang, J.; Paszti, Z.; Wang, F.; Schrauben, J. N.; Tarabara, V. V.; Schmaier, A. H.; Chen, Z. Anal. Bioanal. Chem. 2007, 388, 65–75.
Mefp-3 Conformations at Polymer/Mefp-3 Interfaces method,77to begin the extraction process, the phenol glands (20 g) were allowed to thaw sufficiently to be extruded through a garlic press. They were then mixed with 200 µL of pepstatin A (1 mg/mL in MeOH), 200 µL of leupeptin (1 mg/mL in MeOH), and 200 mL of 5% cold acetic acid in a household blender at high speed for 15 s to reduce the size of the tissue fragments. The mixture was then homogenized with a 30 mL Kontes tissue grinder and centrifuged at 20 000g for 40 min at 4 °C in a Beckman Coulter centrifuge. The pellets were then rehomogenized in 100 mL of 5% cold acetic acid in 8 M urea and centrifuged at 20 000g for 40 min. The supernatant was stirred with 33% ammonium sulfate at 7 °C for 30 min and then centrifuged at 15 000g for 40 min. The supernatant was dialyzed overnight in 4 L of distilled water with 0.7% perchloric acid at 4 °C in 1000 molecular weight cutoff (MWCO) dialysis tubing (Spectra Por), and the contents of the tubing were then centrifuged at 10 000g. The white pellet was redissolved in 2-3 mL of 5% acetic acid in 8 M urea. Immediately before polishing by high performance liquid chromatography (HPLC), the solution was centrifuged at 14 000g for 10 min in an Eppendorf microfuge. The supernatant was injected into an Aquapore C8 column and eluted with a linear gradient of water and acetonitrile in 0.1% trifluoroacetic acid. The eluent was monitored at 280 nm, and the pooled fractions were collected between 20 and 26 min. The eluent was aliquoted into 12 large microcentrifuge tubes (1.5 mL) and freeze-dried overnight, so that the protein could be stored in solid form at -80 °C. Gel electrophoresis has been used to ensure the purity of the Mefp-3 prepared. Calculation of the protein solution concentration was based on extinction coefficients83 at 280 nm with 5500 M cm-1 for tryptophan and 2600 M cm-1 for Dopa. Although all the collected variants were pooled, calculations were based on the amino acids of variant F because the sequence for that variant has been fully determined. The contents of each microcentrifuge tube were dissolved in 1.5 mL of D2O, and a large drop was immediately placed in contact with the solid surface under study. 2.2. Polymer Film Preparation. Deuterated polystyrene (d-PS) and deuterated poly (methyl methacrylate) (d-PMMA) were purchased from Polymer Sources, Inc. A fluorinated polymer, AF2400, was purchased as a 1% solution in FC-75 from DuPont, and a fluorinated adhesion promoter, FC-40, was purchased from 3M. All materials were used as received. Solutions of d-PMMA and d-PS were made by dissolving 2% solid polymer in toluene. The AF-2400 solution was prepared by mixing equal parts of 1% AF2400 solution and FC-40. Polymer films were prepared by placing a few drops of polymer solution onto 1/2′′ fused silica or calcium fluoride prisms (ISP Optics) and spinning on a spin coater (Specialty Coating Systems) at 2000 rpm. Before deposition of the polymer films, the fused silica prisms were heated in a potassium dichromate/ sulfuric acid solution for 20 min before rinsing with deionized water, and the calcium fluoride prisms were scrubbed with soapy water before washing in toluene. The water contact angles for d-PMMA, d-PS, and AF-2400 are 71°, 91°, and 122°, respectively, providing polymer surfaces with a wide range of hydrophobicity. 2.3. SFG Experiments. In a typical SFG experiment, two input beams at frequencies ω1 (in this case, fixed at 532 nm) and ω2 (tunable in the infrared range) are mixed in a medium to generate an output beam at the sum frequency ω ) ω1 + ω2.27–44 If ω2 is scanned over the vibrational resonances of a molecule, then the sum frequency output is resonantly enhanced, producing a vibrational spectrum characteristic of the material. As a second-order nonlinear optical process, the SFG intensity will be zero in a medium with (76) Chen, X.; Wang, J.; Boughton, A. P.; Kristalyn, C. B.; Chen, Z. J. Am. Chem. Soc. 2007, 129, 1420–1427. (77) Papov, V. V.; Diamond, T. V.; Biemann, K.; Waite, J. H. J. Biol. Chem. 1995, 270, 20183–20192. (78) Waite, J. H.; Tanzer, M. L. Science 1981, 212, 1038–1040. (79) Waite, J. H.; Housley, T. J.; Tanzer, M. L. Biochemistry 1985, 24, 5010– 5014. (80) Filpula, D. R.; Lee, S.-M.; Link, R. P.; Strausberg, S. L.; Strausberg, R. L. Biotechnol. Prog. 1990, 6, 171–177. (81) Burzio, L. A.; Waite, J. H. Biochemistry 2000, 39, 11147–11153. (82) Vreeland, V.; Waite, J. H.; Epstein, L. J. Phycol. 1998, 34, 1–8. (83) Gill, S. C.; von Hippel, P. H. Anal. Biochem. 1989, 182, 312–326.
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Figure 1. SFG experimental setup. The bottom side of the prism is coated with the polymer of interest. (a) Input beams overlap on the polymer surface. Output beam originates from the overlap area on the polymer surface and goes to the detector. (b) A drop of protein solution is brought directly into contact with the polymer surface at the input beam overlap area. The output beam is the sum frequency signal of the polymer/solution interface. (c) Small droplet of D2O on the overlap area before contact with protein solution. (d) A drop of protein solution is brought into contact with the polymer film. Protein diffuses through the initial droplet of D2O to adsorb onto the polymer film.
inversion symmetry under the electric-dipole approximation. SFG signals can therefore be detected from the material where the inversion symmetry is broken, usually at a surface or interface. Most bulk materials have inversion symmetry and therefore do not generate SFG signals. Because the input and output beams are polarized, the SFG technique should be able to detect protein orientation or conformation changes at interfaces. In our experiments, the fixed visible 532 nm beam was generated by frequency-doubling the fundamental output pulses of 20 ps pulse width from an EKSPLA Nd:YAG laser. The IR beam, tunable from 1000 to 4000 cm-1, was generated from an EKSPLA optical parametric generation/ amplification and difference frequency system based on LBO and AgGaS2 crystals. The diameters of both beams on the sample were about 0.5 mm. The sum frequency signal was collected by a photomultiplier tube. In this work, SFG spectra with ssp (s ) polarized SFG output, s ) polarized visible input, p ) polarized infrared input) and sps polarization combinations were collected. Because Mefp-3 is a relatively small, hydrophilic protein, it proved necessary to use an optical geometry previously published58 in order to enhance the signal.
3. Results and Discussion 3.1. SFG Studies in the C-H Stretching Region. In this research, deuterated or fluorinated polymers were used to avoid spectral confusion in the C-H stretching frequency region between the polymers and Mefp-3 at the polymer/Mefp-3 solution interfaces. SFG spectra collected from the polymer surfaces in air and polymer surfaces in water in the C-H stretching region do not exhibit any SFG signals, so there was no evidence of hydrocarbon contamination on these surfaces. Likewise, in order to avoid any interference of H2O overlapping the C-H vibrational peaks from Mefp-3, D2O was used to acquire detailed SFG spectra of Mefp-3 at the polymer/solution interface. 3.1.1. Interfacial Ordering of Mefp-3. SFG spectra were collected directly from the polymer/Mefp-3 solution interface with the ssp polarization combination using the experimental geometry shown in Figure 1b. The input beams traveled through the transparent prism substrate and the thin polymer film to reach the polymer/Mefp-3 solution interface, and SFG signals from this interface were detected. Figure 2 displays SFG spectra collected from Mefp-3 at various polymer/solution interfaces. At the d-PMMA/Mefp-3 solution interface (Figure 2a), no protein signals are visible, indicating that the hydrophobic side chain
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Figure 2. SFG spectra in the C-H stretching frequency region collected from the (a) d-PMMA/Mefp-3 solution interface; (b) d-PS/Mefp-3 solution interface; and (c) AF-2400/Mefp-3 solution interface. (d) For comparison, the signal in (c) is multiplied by 5. Protein concentration ) 10 ppm in D2O, ssp polarization.
functional groups of the protein are not strongly ordered at the interface. On the contrary, at the d-PS/Mefp-3 solution interface (Figure 2b), SFG protein signals are clear at ∼2850, ∼2870, ∼2935, ∼3055, and ∼3120 cm-1. The ∼2850, ∼2870, and ∼2935 cm-1 peaks are contributed by the stretching modes of methylene and methyl groups, while the ∼3055 and ∼3120 cm-1 peaks are aromatic C-H stretching signals. The strength of these signals indicates a stronger ordering of the hydrophobic groups here than at the d-PMMA/Mefp-3 solution interface. In contact with AF-2400 (Figure 1c), the same peaks are weaker but still visible, except that the spectral features are different. SFG has been used to study interfaces between solid contacting media and protein solutions in situ in the C-H stretching region.53,54 SFG C-H stretching signals are mostly contributed by protein side chain groups. In our previous studies, we reported that different protein side chain structures have been detected from varied solid/liquid interfaces for a variety of proteins. For example, for blood proteins albumin and fibrinogen,53,54,71 we found that no SFG C-H signals were detected from the fused silica/protein solution interface. We believe that this is due to the fact that the hydrophobic side chains fold into the protein because both water and fused silica contacting media are hydrophilic. The interactions between hydrophobic side chains and such hydrophilic environments are not favorable, and thus, they tend to stay inside the protein and do not exhibit net ordering at the interface, resulting in no detectable SFG signals. However, at hydrophobic contacting media/protein solution interfaces, protein hydrophobic side chains prefer to face to the hydrophobic contacting media, due to the favorable interactions, thus generating strong SFG signals. Here, for Mefp-3, we observed results similar to those for albumin and fibrinogen using SFG. PMMA is relatively hydrophilic, and at the PMMA/Mefp-3 solution interface C-H side chains are not likely to adopt a preferred orientation and no SFG signal was observed. However, PS and AF-2400 have much more hydrophobic surfaces, inducing more ordered orientations of hydrophobic side chain groups of Mefp-3 at these two interfaces. This shows clearly that Mefp-3 adopts different structures after coming in contact with different surfaces. 3.1.2. Interfacial BehaVior of Certain Amino Acid Residues of Mefp-3. The primary structure for one Mefp-3 family member, Mefp-3F, has been completely determined. If this variant can be considered broadly representative of the pool of Mefp-3 variants used in these experiments, then the SFG peaks can be correlated to the interfacial behavior of the most hydrophobic amino acids of the protein. The hydrophobicity of amino acids has been studied
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Figure 3. SFG spectral signals in the C-H stretching frequency region collected from the d-PS/Mefp-3 solution interface and AF-2400/Mefp-3 solution interface. Protein concentration ) 10 ppm in D2O, sps polarization.
in detail before.84 Variant F is known to contain one alanine, three prolines, three tryptophans, nine tyrosines, and ten Dopas; these would correspond to the methyl, methylene, and phenyl signals visible on the SFG spectra. The protein film appears to be less well-ordered at the solution/AF-2400 interface, although the same vibrational peaks are detected here at the solution/d-PS solution interface. Vibrational spectra of hydrophobic amino acid residues such as alanine, proline, tryptophan, tyrosine, and Dopa have been published.85–91 The methyl symmetric stretching signal of alanine is at 2870 cm-1.85–87 Proline CH2 symmetric stretching peaks are present at 2865, 2885, and 2935 cm-1.88 For tryptophan, the CH2 stretching modes have signals at 2870 and 2935 cm-1, and C-H stretching signals of the aromatic group are at a little bit higher frequency (>3000 cm-1).89 The vibrational peaks for CH2 in tyrosine are centered at 2827 and 2943 cm-1, and aromatic C-H stretching modes are >3000 cm-1.90 Similarly, the Dopa also has aromatic C-H stretching at >3000 cm-1. Its CH2 stretching signals are at 2856 and 2928 cm-1. At the AF-2400/Mefp-3 solution interface (Figure 2c and d), the peak at 2870 cm-1 is relatively stronger compared to the 2935 cm-1 peak. This peak should be contributed by the methyl group in alanine and the CH2 groups in proline and tryptophan. The peak at 2935 cm-1 should be due to the CH2 stretching of proline, tryptophan, tyrosine, or Dopa, and the peak at 3055 cm-1 must be contributed by the aromatic groups in tryptophan, tyrosine, and Dopa. At the solution/d-PS interface (Figure 2b), similar signals have been detected, but with different relative intensities. Similarly, we believe that the strong peak at 2935 cm-1 must be due to the proline, tryptophan, tyrosine, or Dopa CH2 groups, and the 2870 cm-1 can be contributed by alanine CH3 and/or proline/tryptophan CH2 groups. We also collected sps SFG spectra from the d-PS/Mefp-3 solution and AF-2400/ Mefp-3 solution interface (Figure 3). The dominating peaks in these spectra are contributed by the methyl asymmetric stretching at 2960 cm-1. This shows that alanine is present at both interfaces. Since there is only one alanine residue in variant F of Mefp-3 and likely one or very few in the other pooled variants, we believe that SFG can detect signals from a single amino acid in a protein at the solid/liquid interface. Although no sps signals were detected (84) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246, 2211–2217. (85) Rosadoa, M. T. S.; Duarteb, M. L. R. S.; Faustoc, R. J. Mol. Struct. 1997, 410-411, 343–348. (86) Cao, X.; Fischer, G. Chem. Phys. 2000, 255, 195–204. (87) Cao, X.; Fischer, G. Spectrochim. Acta, Part A 1999, 55, 2329–2342. (88) Reva, I. D.; Stepanian, S. G.; Plokhotnichenko, A. M.; Radchenko, E. D.; Sheina, G. G.; Blagoi, Y. P. J. Mol. Struct. 1994, 318, 1–13. (89) Cao, X.; Fischer, G. J. Phys. Chem. A 1999, 103, 9995–10003. (90) Inomata, Y.; Inomata, T.; Moriwaki, T. Bull. Chem. Soc. Jpn. 1974, 47, 818–824. (91) Petoral, R. M., Jr.; Uvdal, K. J. Phys. Chem. B 2003, 107, 13396–13402.
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Figure 4. SFG spectra in the C-H stretching frequency region collected from the (a) d-PS/Mefp-3 solution interface (initial) and (b) d-PS/Mefp-3 solution interface (stable). Protein concentration ) 10 ppm in D2O, ssp polarization.
Figure 5. SFG spectra in the C-H stretching frequency region collected from the (a) AF-2400/Mefp-3 solution interface (initial) and (b) AF2400/Mefp-3 solution interface (stable). Protein concentration ) 10 ppm in D2O, ssp polarization.
for CH2 groups at the AF-2400/Mefp-3 solution interface, they were detected at the d-PS/Mefp-3 solution interface. Stronger CH2 signals and aromatic C-H signals were detected at the d-PS/Mefp-3 solution interface, which is reasonable because the phenyl group on the PS surface may have more favored interactions with proline, tryptophan, tyrosine, and Dopa, which have ring structures. The overall much weaker intensity may represent a more broad orientation distribution or disordered adsorption of Mefp-3 at the AF-2400 interface, which will be discussed in more detail below. 3.1.3. Time-Dependent Interfacial BehaVior of Mefp-3. We have also investigated the time-dependent behavior of interfacial Mefp-3. One possible explanation was that the interfacial protein spectrum changed over time. The SFG C-H signals from Mefp-3 appear to be relatively stable over time at the solution/d-PS interface (Figure 4b), but the signals underwent a considerable change over time at the solution/AF-2400 interface (Figure 5b). This shows that somehow Mefp-3 at the solution/d-PS interface quickly reached an equilibrated structure, while Mefp-3 at the solution/AF-2400 interface reaches equilibrium more slowly. For the latter case, the initially very strong CH2(s) and CH3(s) peaks quickly shrank, while the phenyl peak grew, until the spectrum adopted the peak ratios found in Figure 5b. It may be that, in the course of the experiment, before the drop of protein was brought into contact with the polymer (Figure 1b), the protein film at the surface of the drop adopted a specific conformation at the solution/air interface. Subsequent contact with the fluorinated polymer then induced a slow conformational change seen in Figure 5. A much weaker change was observed in Figure 4, probably because the interaction between d-PS and Mefp-3 is so strong, due to the ring-ring interactions, that any significant
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Figure 6. SFG spectra in the C-H stretching frequency region collected from the (a) d-PS/Mefp-3 solution interface and (b) AF-2400/Mefp-3 solution interface. Protein concentration ) 10 ppm in D2O, ssp polarization.
conformational change would be too fast to track by SFG, and the result is a more strongly ordered protein film at the interface. To further investigate the effect of adsorbed Mefp-3 at the solution/polymer interfaces and confirm our hypothesis for the time-dependent SFG signal change above, the sample handling method was modified. A small drop of D2O was placed on the polymer surface (Figure 1c) before contact with the protein solution was made. The protein solution was then brought into contact at the spot where the original drop of D2O was placed (Figure 1d). This method provided only a limited change in the results for the solution/d-PS interface (Figure 6a), which was very consistent. However, from Figure 6b, it is clear that at the AF-2400 interface the protein immediately and consistently adopted the final conformation as shown in Figure 5b, without exhibiting an initial solution/air conformation. Previous SFG studies indicated that the PS surface is dominated by phenyl groups.32,36 When the PS contacts the Mefp-3 solution, because of the favorable interactions between phenyl groups on the PS surface (in this case, deuterated phenyl group on the d-PS surface) and amino acid residues with ring structures (e.g., proline, tryptophan, tyrosine, and Dopa) in Mefp-3, the aromatic and CH2 groups (along with some methyl groups) segregate to the surface immediately, no matter whether methyl or other groups first contact the PS surface. However, on the AF-2400 surface, dominated by fluorinated groups that can interact with both CH3 and aromatic groups, some time is required for aromatic amino acids to dominate the interface if methyl groups are initially dominant there. 3.2. SFG Studies in the Amide Range. It is well-known that protein amide I vibrational signals can be used to deduce protein secondary structural information. We demonstrated the feasibility to detect SFG amide I signals from polymer/protein solution interfaces in situ.58 Furthermore, we showed that SFG signals can be used to differentiate different secondary structures such as R-helices and β-sheets because such secondary structures have their own characteristics.67 Therefore, SFG should be able to probe secondary structural information of proteins at interfaces. Mefp-3 is a small protein with little known secondary structure in solution. However, spectral information in the amide range may contribute to a picture of its adsorption behavior. SFG spectra were collected from interfaces between Mefp-3 solution and the various polymers studied above in the amide I spectral region, and these spectra are shown in Figure 7. Corresponding to the conformational behavior exhibited in the C-H stretching range, no SFG amide I signal is detected at the solution/d-PMMA interface (Figure 7a), showing that, at the PMMA/Mefp-3 solution interface, the backbone of Mefp-3 also
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Figure 7. SFG spectra in the amide I frequency region collected from the (a) d-PMMA/Mefp-3 solution interface; (b) d-PS/Mefp-3 solution interface; and (c) AF-2400/Mefp-3 solution interface. Protein concentration ) 10 ppm in D2O, ssp polarization.
adopts a random structure. The peak at about 1730 cm-1 is due to the CdO stretching mode of the PMMA surface. A clear but weak SFG amide I signal was detected at the solution/d-PS interface (Figure 7b) at 1665 cm-1, which may correspond to some ordering of Mefp-3 secondary structures. An even weaker, broad amide I signal was detected at the solution/AF-2400 interface (Figure 7c). This shows that at the interface the Mefp-3 secondary structure ordering is less compared to the secondary structure at the solution/d-PS interface. These results appear to correspond to the SFG spectra in the CH range, with a maximum of interfacial ordering of Mefp-3 at the d-PS interface, no order detected at the d-PMMA interface, and an intermediate degree of order at the AF-2400 interface. 3.3. Further Discussion. Substrate surface chemistry can strongly influence the structure of an adsorbed protein. The adhesive pads of Mytilus edulis exhibit stronger attachment to polar surfaces than nonpolar surfaces. The area of the plaque is related to the surface energy of the substrate,3 with the size of the pad increasing with decreasing polarity of the substrate. The protein can more easily displace water and spread out on a hydrophobic surface.92 In fact, a significant driving force for protein adsorption on any surface is the entropy increase when water is displaced from the substrate surface, decreasing the Gibbs free energy for the protein adsorption process.23,93,94 It is interesting to note that the Mytilus edulis primer proteins are the most extensively hydroxylated of all the proteins in the adhesive plaque. This may allow them to chemically behave in a way similar to water and therefore easily displace water molecules at most interfaces. In comparing the adsorption behavior of Mefp-3 on the three polymer surfaces, the SFG results are compatible with earlier studies done on Mefp-1 using different methods. It has been shown by atomic force microscopy (AFM)23 that a film of Mefp-1 and Mefp-2 adsorbed onto PS was stabilized by functional group interactions (Dopa/phenyl π-π) that prevented disruption of the protein layer upon dehydration. This would account for the strong, stable SFG signal seen for Mefp-3 at the d-PS/protein solution interface. As we showed above, such signals are dominated by the contributions from amino acid residues with ring structures. Mytilus edulis byssus pads form larger contact areas at more hydrophobic surfaces.95 It has also been shown that Mefp-1 adopts an extended, hydrogel-like structure on a hydrophobic surface.96 Hydration of the polar groups on the mussel adhesive proteins may be governed by its interactions with the substrate, and this (92) Crisp, D. J.; Walker, G.; Young, G. A.; Yule, A. B. J. Colloid Interface Sci. 1985, 104, 40–50. (93) Norde, W. Cells Mater. 1995, 5, 97–112. (94) Norde, W. Macromol. Symp. 1996, 103, 5–18.
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is supported by the SFG spectra. At the d-PMMA interface where carbonyl groups from the polymer would be present, adhesion can be dominated by hydrogen bonding involving many of the various hydrophilic amino acids of the protein, leaving very few ordered hydrophobic amino acids at the interface to contribute signals to the spectrum. At the AF-2400 interface, which is the most hydrophobic, there are no particularly strong interactions and the protein film is extensively hydrated, reducing the contact of the hydrophobic amino acids with the polymer at the interface. This is because the surface dominating fluorinated groups of AF-2400 do not interact as favorably with CH3, CH2, and aromatic C-H groups on hydrophobic amino acid residues. Therefore, the overall SFG signal intensity is much weaker compared to the d-PS/Mefp-3 solution interface, and the hydrophobic amino acid residues must be much more disordered at the AF-2400/Mefp-3 solution interface. It is interesting to note that the terminal amino acids of Mefp-3F contribute signals at the very hydrophobic AF-2400 interface. The conformational freedom of these groups further substantiates the weak contact that the adsorbates make at the AF-2400 interface. It has been proposed that the hydrophobic N-terminal half of Mefp-3 (with the alanine and several Dopas) tends to mix with the bulk of the plaque proteins in order to cross-link with them, while the other, more hydrophilic half of the protein tends to make contact with surfaces for attachment.97 However, our results show that, at the hydrophobic surface, the alanine end of the protein is clearly in contact with and wellordered at the interface. This difference would suggest that hydrophobic surfaces may induce a different Dopa cross-linking pattern from that found at hydrophilic surfaces. In this work, we demonstrated that SFG can elucidate structural differences of Mefp-3 in contact with different polymers in situ. We believe that this is the first interfacial spectroscopic study done on the primer protein, Mefp-3, which should be the most relevant to the adsorption behavior of the adhesive plaque. It was also the first detection of amide range signals for this protein, and the first example of an interfacial SFG study of conformational behavior of a very few specific hydrophobic amino acids. In the case of variant F and many similar variants, an individual alanine can be singled out on a complete protein. This research indicates that the ordering of hydrophobic side chains of Mefp-3 can be altered at different polymer/protein solution interfaces, due to the varied interfacial interactions. Some specific interactions between polymer surface functional groups and certain amino acid residues on Mefp-3 have been probed.
4. Conclusion SFG studies indicate that Mefp-3 adopts varied conformations at various polymer/Mefp-3 solution interfaces. At the hydrophilic polymer surface, no SFG signals can be detected in the C-H stretching or amide I signal regions. This shows that Mefp-3 adopts more or less a random structure for hydrophobic side chains and secondary structures. On hydrophobic surfaces, strong SFG signals were detected in both the C-H stretching and amide I regions, indicating ordered adsorption. We are currently examining Mefp-3 conformations at various poly(dimethyl siloxane) (PDMS) interfaces using SFG. These PDMS materials are being developed as antifouling or fouling release coatings for marine vessels. Such studies will have a more direct impact on the rational design and development of (95) Aldred, N.; Ista, L. K.; Callow, M. E.; Callow, J. A.; Lopez, G. P.; Clare, A. S. J. R. Soc. Interface 2006, 3, 37–43. (96) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796–5804. (97) Warner, S. C.; Waite, J. H. Mar. Biol. 1999, 134, 729–734.
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polymer coatings with improved properties. In this paper, the detailed conformation of Mefp-3 at the interface has not been able to be deduced. We have recently studied the detailed conformation and orientation of fibrinogen and Gβγ using SFG according to their crystal structures.98,99 It is difficult to do so for Mefp-3 due to the lack of a crystal structure. We are attempting to derive more detailed structural information on Mefp-3 from SFG studies involving the prediction of a protein structure from its sequence using various available software packages. Such results will be reported in a separate publication. In the future, synthetic Mefp-3 with certain isotope labeled amino acids will (98) Wang, J.; Lee, S. H.; Chen, Z. J. Phys. Chem. B 2008, 112, 2281–2290. (99) Chen, X.; Boughton, A. P.; Tesmer, J. J. G.; Chen, Z. J. Am. Chem. Soc. 2007, 129, 12658–12659.
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be studied using SFG, which should lead to a more detailed structural determination of Mefp-3 at various interfaces. We believe that continued success in studying Mefp-3 at various interfaces should provide a more detailed understanding regarding molecular mechanisms of biofouling and bioadhesion. In the future, SFG studies on individual variants of Mefp-3 at different interfaces should also be carried out to elucidate the role of the different variants. Acknowledgment. This research is supported by the Office of Naval Research (N00014-02-1-0832). We want to thank Professor Herbert Waite for his advice and help in this research. LA800138X
