Probing α-Helical and β-Sheet Structures of ... - ACS Publications

Theoretical Sum Frequency Generation Spectroscopy of Peptides. Joshua K. Carr , Lu .... The Journal of Physical Chemistry B 0 (proofing),. Abstract | ...
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Probing r-Helical and β-Sheet Structures of Peptides at Solid/Liquid Interfaces with SFG Xiaoyun Chen, Jie Wang, Jason J. Sniadecki, Mark A. Even, and Zhan Chen* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109 Received January 6, 2005 We demonstrated that sum frequency generation (SFG) vibrational spectroscopy can distinguish different secondary structures of proteins or peptides adsorbed at solid/liquid interfaces. The SFG spectrum for tachyplesin I at the polystyrene (PS)/solution interface has a fingerprint peak corresponding to the B1/B3 mode of the antiparallel β-sheet. This peak disappeared upon the addition of dithiothreitol, which can disrupt the β-sheet structure. The SFG spectrum indicative of the MSI594 R-helical structure was observed at the PS/MSI594 solution interface. This research validates SFG as a powerful technique for revealing detailed secondary structures of interfacial proteins and peptides.

In this letter, we demonstrate the feasibility of using sum frequency generation (SFG) vibrational spectroscopy to distinguish between different secondary structures of polypeptides such as R-helices and β-sheets at solid/liquid interfaces. The molecular structures of proteins and polypeptides adsorbed at solid/solution interfaces play important roles in many applications such as biomaterials, biosensors, marine anti-biofouling coatings, antimicrobial peptides, and the food industry.1 In the last few decades, substantial progress has been made to achieve a molecular level understanding of protein or polypeptide structures in bulk environments such as in solution or crystal form, owing to the improvements of modern analytical techniques, especially in X-ray crystallography and NMR spectroscopy. However, the elucidation of the molecular structures of proteins and polypeptides at interfaces is still difficult. Recently, SFG has been developed into a powerful technique for studying biological molecules.2-5 Along with other research groups, we have demonstrated the feasibility of using SFG to observe the hydrophobic side chain conformations of proteins at various solid/liquid interfaces through studies of C-H stretching modes3 and successfully detected amide I signals from interfacial peptides and proteins.4,5 The application of SFG to study interfacial protein structures is important but still at an early stage. Correlations between SFG spectra, especially in the amide I region, and protein interfacial structures * To whom correspondence should be addressed. E-mail: [email protected]. Fax: 734-647-4685. (1) (a) Horbett, T. A., Brash, J. L., Eds. Proteins at Interfaces II, Fundamentals and Applications; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (b) Wynne, K. J.; Guard, H. Nav. Res. Rev. 1997, 49, 1-3. (c) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110-115. (d) Albers, W. M.; Vikholm, I.; Viitala, T.; Peltonen, J. In Handbook of Surfaces and Interfaces of Materials; Nalwa, H. S., Ed.; Academic Press: San Diego, CA, 2001; pp 1-31. (e) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233-244. (2) (a) Ye, S.; Noda, H.; Nishida, T.; Morita, S.; Osawa, M. Langmuir 2004, 20, 357-365. (b) Doyle, A.; Fick, J.; Himmelhaus, M.; Eck, W.; Graziani, I.; Prudovsky, I.; Grunze, M.; Maciag, T.; Neivandt, D. Langmuir 2004, 20, 8961-8965. (c) Liu, J.; Conboy, J. J. Am. Chem. Soc. 2004, 126, 8894-8895. (3) (a) 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. (b) Kim, J.; Somorjai, G. A. J. Am. Chem. Soc. 2003, 125, 3150-3158. (c) Wang, J.; Buck, S. M.; Even, M. A.; Chen, Z. J. Am. Chem. Soc. 2002, 124, 13302-13305. (d) Dreesen, L.; Humbert, C.; Sartenaer, Y.; Caudano, Y.; Volcke, C.; Mani, A. A.; Peremans, A.; Thiry, P. A.; Hanique, S.; Frere, J.-M. Langmuir 2004, 20, 7201-7207. (4) Wang, J.; Even, M. A.; Chen, X.; Schmaier, A. H.; Waite, J. H.; Chen, Z. J. Am. Chem. Soc. 2003, 125, 9914-9915. (5) Knoesen, A.; Pakalnis, S.; Wang, M.; Wise, W. D.; Lee, N.; Frank, C. W. IEEE J. Sel. Top. Quantum Electron. 2004, 10, 1154-1163.

Figure 1. (right) Tachyplesin I with -S-S- representing the intrastrand disulfide bonds that stabilize the β-sheet structure. (left) Helical wheel diagram of MSI594.

have not yet been illustrated in detail. The purpose of this paper is to show the feasibility of using SFG to recognize and to study different secondary structures, providing a basis for future detailed elucidation of the molecular structures of proteins and peptides at interfaces. In this research, we used two model peptides with wellcharacterized structures as model systems in the SFG experiments: MSI594 and tachyplesin I (Figure 1). MSI594 consists of 24 amino acid residues with a net charge of +6. As a peptide in the magainin family, it adopts an R-helical structure when interacting with a membrane surface.6 Tachyplesin I is composed of 17 amino acid residues and has a rigid antiparallel β-sheet structure connected by a β-turn and two intrastrand disulfide bonds both in solution and on membrane surfaces.7,8 We believe that the conformations of these two peptides at the polystyrene (PS)/solution interfaces should not be very different from those on membrane surfaces, due to the similar asymmetric environment where the peptides are located. We can confirm this from both our SFG and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) results. The relatively small size of these two peptides compared to protein molecules makes it possible to deconvolute the complicated amide I SFG spectra. We believe such an SFG study on model peptides will serve as a basis for future SFG research on the detailed secondary structures of interfacial proteins. The details of our SFG experiments have been described previously.9 A total reflection geometry was used with a (6) (a) Matsuzaki, K. Biochim. Biophys. Acta 1998, 1376, 391-400. (b) Matsuzaki, K. Biochim. Biophys. Acta 1999, 1462, 1-10. (c) Personal communication with the Ramamoorthy Group, University of Michigan. (7) Matsuzaki, K.; Nakayama, M.; Fukui, M.; Otaka, A.; Funakoshi, S.; Fujii, N.; Bessho, K.; Miyajima, K. Biochemistry 1993, 32, 1170411710. (8) (a) Laederach, A.; Andreotti, A. H.; Fulton, D. B. Biochemistry 2002, 41, 12359-12368. (b) Park, N. G.; Lee S.; Oishi O.; Aoyagi H.; Iwanaga S.; Yamashita S.; Ohno, M. Biochemistry 1992, 31, 1224112247.

10.1021/la050048w CCC: $30.25 © 2005 American Chemical Society Published on Web 03/01/2005

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Figure 3. (a) SFG and (b) ATR-FTIR spectra and fitting results (a nonresonant background of -6 was used for SFG spectral fitting) for 0.1 mg/mL MSI594 adsorbed at the solution/PS interface, with the squares representing the actual spectra, the dotted lines representing the fitted spectra, and the solid lines representing the component peaks used to fit the spectra.

Figure 2. Spectra and fitting results (a nonresonant background of -4 was used for SFG spectral fitting) for 0.1 mg/mL tachyplesin I adsorbed at the solution/PS interface, with the squares representing the actual spectra, the dotted lines representing the fitted spectra, and the solid lines representing the component peaks used to fit the spectra: (a and b) SFG spectra before and after contacting 10 mM DTT; (c) ATR-FTIR spectrum.

CaF2 prism spin-coated with a PS film.4 All SFG spectra shown here were collected with an ssp (s-polarized SFG signal, s-polarized visible, and p-polarized IR beam) polarization combination. All the other experimental procedures regarding sample preparation, ATR-FTIR, and SFG data analysis can be found in the Supporting Information. Figure 2a shows the amide I SFG spectrum collected from tachyplesin I adsorbed at the PS/solution interface and the fitting result. Two peaks can be easily identified in this range, a strong peak around 1664 cm-1 and a weaker peak at 1688 cm-1. The peak fitting results identify an additional peak at 1645 cm-1 and several minor peaks at lower wavenumbers. Figure 2b displays the SFG spectrum collected from the PS/solution interface after the addition of dithiothreitol (DTT) into the solution. Apparently, the 1688 cm-1 peak disappeared, while the bands between 1640 and 1670 cm-1 remained. Another adsorption experiment using tachyplesin I solution, treated with DTT first and then followed by contact with PS, led to a very similar SFG spectrum collected from the PS/solution interface. As has been discussed in many previously published papers, amide signals at 1688 and 1633 cm-1 can be ascribed to the B1/B3 and B2 vibrational modes of antiparallel β-sheets, respectively.10,11 The 1664 and 1645 cm-1 peaks in the tachyplesin I SFG spectra are due to turns, (9) Wang, J.; Chen, C.; Buck, S. M.; Chen, Z. J. Phys. Chem. B 2001, 105, 12118-12125. (10) Keiderling, T. A. J. Am. Chem. Soc. 2003, 125, 7562-7574.

random structures, or a combination of them.11,12 The 1645 cm-1 peak may also have some contribution from the B2 mode of β-sheets. More details regarding the assignments for these peaks are under current investigation. Below, we will focus on the SFG band around 1688 cm-1. The disappearance of the 1688 cm-1 peak in the SFG spectrum after the addition of DTT confirms that this peak is generated by the β-sheet structure of tachyplesin I at the interface. The two disulfide bonds are essential for tachyplesin I to maintain its β-sheet structure.7 DTT is often used to elucidate the function of disulfide bonds in proteins/peptides with its ability to reduce the disulfide bonds.13 We believe that the disappearance of the 1688 cm-1 peak is due to the loss of antiparallel β-sheet structure as a consequence of the cleavage of the two intramolecular disulfide bonds. This shows clearly that the 1688 cm-1 peak is contributed by the β-sheet structure at the interface. For the purpose of further confirmation and comparison, an ATR-FTIR spectrum has also been collected from tachyplesin I at the PS/solution interface with a dominant peak at 1633 cm-1, as shown in Figure 2c. The relative peak heights at 1633, 1658, 1671, and 1690 cm-1 (from the B2 mode, random structures, β-turns, and B1/B3 modes, respectively) agree very well with the antiparallel β-sheet conformation of interfacial tachyplesin I.7,10,12 The B1/B3 and B2 modes of normal bulk β-sheet structures are both IR and Raman active, and should be SFG active.11 It is interesting to note that no strong SFG peak is identified around the 1633 cm-1 range. We believe that the difference between the bands of the β-sheet in tachyplesin I detected in the SFG and ATR-FTIR spectra are due to the different selection rules of these two spectroscopic techniques and different dependence of the peak intensities on the orientation of the interfacial peptides. Our research here demonstrates very clearly that SFG can detect amide I signals from β-sheet structures of interfacial peptides. Shown in Figure 3a is the amide I SFG signal collected from MSI594 at the PS/solution interface. The main amide signal is centered at 1650 cm-1, showing that the R-helical structure of MSI594 dominates at the PS/solution inter(11) Krimm, S.; Bandekar, J. Adv. Protein Chem. 1986, 38, 18138364. Hilario, J.; Kubelka, J.; Barth, A.; Zscherp, C. Q. Rev. Biophys. 2002, 35, 369-430. (12) Vass, E.; Hollosi, M.; Besson, F.; Buchet, R. Chem. Rev. 2003, 103, 1917-1954. (13) Li, Y.-J.; Rothwarf, D. M.; Scheraga, H. A. J. Am. Chem. Soc. 1998, 120, 2668-2669.

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face.11 The SFG spectrum can be fitted quite well using a single vibrational peak and a nonresonant background, as shown in Figure 3a. The ATR-FTIR spectrum in Figure 3b also confirms that MSI594 does adopt an R-helical structure at the PS/peptide solution interface. A more detailed interpretation about fitting can be found in the Supporting Information. SFG has several advantages over conventional ATRFTIR. In this work, meticulous subtraction of the background signal, especially the water bending signal, is required to achieve ATR-FTIR spectra of decent signalto-noise ratio. Usually, such background signals are many times stronger, and thus, serious errors can occur in the spectral subtraction. SFG has the advantage of detecting spectra from interfacial molecules directly, without the necessity to subtract background signals and is thus less prone to error. Another important feature is that SFG can provide different structural information than the ATRFTIR technique. For instance, to determine the orientation distribution of a secondary structure, for example, the R-helical structure of MSI594 at the interface, ATR-FTIR can measure the average cos2 θ, while SFG can measure the average cos θ and the average cos3 θ, with θ being the angle between the helix axis and the surface normal. Additional surface structural parameters can also be measured by SFG.14 Combining ATR-FTIR and SFG, we can acquire more accurate information about the structures of proteins and peptides at interfaces. The structures of antimicrobial peptides in membranes have been extensively studied. However, some uncertainties still remain in such studies. For example, NMR has

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substantially progressed our understanding of peptidemembrane interactions, but the samples used in NMR studies are composed of many layers of lipids with peptides, instead of peptides in one bilayer, which is the real situation in a biological system.15 In addition, the absolute orientation of a peptide, for example, whether a helix points up or down, cannot be determined by NMR studies. SFG can be used to study peptide structure in one lipid bilayer and can probe the absolute orientation of peptides. Conclusion This paper has illustrated the feasibility to distinguish between various secondary structures of peptides at solid/ liquid interfaces in situ using SFG. We demonstrated that SFG amide I signals can be detected from R-helix and β-sheet structures at the interface. This research validates SFG as a powerful technique for revealing the detailed secondary structures of interfacial proteins and peptides. Detailed orientation calculations for the peptides at polymer/solution interfaces are currently under investigation. Acknowledgment. This research is supported by the Beckman Foundation and Office of Naval Research. We thank the Ramamoorthy group for providing the MSI594 samples. We also appreciate our inspiring discussion with the Krimm group on the normal modes of β-sheets. Supporting Information Available: Experimental procedures regarding sample preparation, ATR-FTIR, and SFG data analysis. This material is available free of charge via the Internet at http://pubs.acs.org. LA050048W

(14) Wei, X.; Hong, S.-C.; Zhuang, X.; Goto, T.; Shen, Y. R. Phys. Rev. E 2000, 62, 5160-5172.

(15) Bechinger, B. Biochim. Biophys. Acta 1999, 1462, 157-183.