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Cytochrome c at Model Membrane Surfaces: Exploration via Second Harmonic Generation-Circular Dichroism and Surface-Enhanced Resonance Raman Spectroscopy T. P. Petralli-Mallow,*,† A. L. Plant,† M. L. Lewis,‡ and J. M. Hicks§ Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, Department of Chemistry, University of Denver, Denver, Colorado 80208, and Department of Chemistry, Georgetown University, Washington, D.C. 20057 Received October 7, 1999. In Final Form: March 31, 2000 The novel nonlinear optical method of second harmonic generation-circular dichroism (SHG-CD) has been used to follow the adsorption and redox properties of a peripheral membrane protein, horse heart cytochrome c, adsorbed at several model membrane surfaces. The SHG-CD response is shown to be affected by the oxidation state of the heme within surface-adsorbed cytochrome c, as is consistent with the sensitivity of SHG to the chirality of the heme. SHG-CD measurements show that adsorbed cytochrome c is reducible by ascorbate at alkanethiol surfaces, but not at phospholipid/alkanethiol hybrid bilayer membranes (HBMs). The adsorption of cytochrome c at the model membrane surfaces was verified by surface plasmon resonance. Surface-enhanced resonance Raman measurements show that cytochrome c adsorbed on a hybrid bilayer membrane retains the Fe-heme conformation associated with solution-phase cytochrome c and is reducible by applying potential to the supporting electrode. The inability of ascorbic acid to reduce cytochrome c associated with the HBM is attributed not to a change in its redox potential, but rather to the nature of the interaction of cytochrome c with the HBM.
Introduction There are many unanswered questions about the structures and functions of the molecules comprising biological membranes. In this paper, we apply the surfacesensitive optical techniques of second harmonic generation-circular dichroism (SHG-CD) and surface-enhanced resonance Raman (SERRS) to an in situ study of a peripheral membrane protein, cytochrome c, adsorbed at membrane mimetic surfaces. Cytochrome c is an especially interesting redox species due to its membrane-associated activity and its biological role in electron transport. As a result, cytochrome c has been the subject of many biophysical studies.1,2 Cytochrome c has been shown to adsorb to hydrophobic and hydrophilic alkanethiol surfaces and at silanized fused silica.3 At carboxylic acid-terminated self-assembled monolayers (SAMs), cytochrome c forms a stable and electroactive layer that is reducible by applying an electrode potential4 or by the addition of a reducing agent to solution.5 Cytochrome c has also been studied on planar lipid surfaces2,6-9 and liposomes.2,10-12 Hybrid bilayer membranes (HBMs) have been shown to have many properties of cell and model membranes, * To whom correspondence should be addressed. E-mail:
[email protected]. † National Institute of Standards and Technology. ‡ University of Denver. § Georgetown University. (1) Sankaram, M. B.; Marsh, D. In Protein-Lipid Interactions; Watts, A, Ed.; Elsevier Science Publishers: New York, 1993; pp 127-161. (2) Nicholls, P. Biochim. Biophys. Acta 1974, 346, 261-310. (3) Edminston, P. L.; Lee, J. E.; Cheng; S.-S.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 560-570. (4) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (5) Maeda, Y.; Yamamoto, H.; Kitano, H. J. Phys. Chem. 1995, 99, 4837-4841. (6) Steinmann, A.; Lauger, P. J. Membr. Biol. 1971, 4, 74-86. (7) Sui, S.-F.; Wu, H.; Sheng, J.; Guo, Y. J. Biochem. 1994, 115, 10531057. (8) Pachence, J. M.; Amador, S.; Maniara, G.; Vanderkooi, J.; Dutton, P. L.; Blasie, J. K. Biophys. J. 1990, 58, 379-389.
with added advantages in ruggedness, extended stability, and ease of preparation.13,14 HBMs are electricallyaddressable using the underlying gold film as a working electrode; electrochemical measurements have been used to study the interactions of adsorbates with HBMs.13 The nonlinear optical technique second harmonic generation (SHG) exhibits extreme surface specificity, submonolayer sensitivity, and the ability to probe buried aqueous interfaces.15,16 In addition, SHG-CD is sensitive to chirality.17-20 As of yet, SHG and SHG-CD have had only a few applications to biophysics. Previous SHG-CD applications to biomolecules include the study of a chiral peptide, Boc-trp-trp, at the air/water interface,21 and the study of bacteriorhodopsin, an integral membrane protein involved in proton pumping.22 Since SHG and SHG-CD can be used to probe dielectric interfaces, it is possible that such nonlinear optical techniques may be useful for in situ studies of native biological membranes. (9) Kozarac, Z.; Dhathathreyan, A.; Mobius, D. FEBS Lett. 1988, 229, 372-376. (10) Hildebrandt, P.; Heimburg, T.; Marsh, D. Eur. Biophys. J. 1990, 18, 193-201. (11) Muga, A.; Mantasch, H. H.; Surewicz, W. K. Biochemistry 1991, 30, 0, 7219-7224. (12) Cheddar, G.; Tollin, G. Arch. Biochem. Biophys. 1992, 294, 188192. (13) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126-1133. (14) Plant, A. L.; Brigham-Burke, M.; Petrella, E. C.; O’Shannessy, D. J. Anal. Biochem. 1995, 226, 342-348. (15) Corn, R. M.; Higgins, D. A. Chem. Rev. 1994, 94, 107-125. (16) Shen, Y. R. Nature 1989, 337, 519-525. (17) Hicks, J. M.; Petralli-Mallow, T. Appl. Phys. B 1998, 68, 589593. (18) Hicks, J. M.; T.Petralli-Mallow; Byers, J. D. Faraday Discuss. 1994, 99, 341-357. (19) Kauranen, M.; Verbiest, T.; Persoons, A. J. Mod. Opt. 1998, 45, 403-423. (20) Kauranen, M.; Verbiest, T.; Maki, J. J.; Persoons, A. J. Chem. Phys. 1994, 101, 8193-8199. (21) Crawford, M. J.; Haslam, S.; Probert, J. M.; Gruzdkov, Y. A.; Frey, J. G. Chem. Phys. Lett. 1994, 229, 260-264. (22) Verbiest, T.; Kauranen, M.; Persoons, A. J. Am. Chem. Soc. 1994, 116, 9203-9205.
10.1021/la9913250 CCC: $19.00 © 2000 American Chemical Society Published on Web 06/13/2000
Cytochrome c at Model Membrane Surfaces
SHG-CD has been shown to be sensitive to the chirality, and thus the structure, of chiral molecules at surfaces.17-20 Electric-dipole allowed SHG-CD requires that the surfaceadsorbed molecule have a chromophore that is chiral and has a one- or two-photon electronic resonance within the range of incident laser wavelengths.23 In the case of cytochrome c, the heme group is two-photon resonant with the laser light in our experiments. If the heme is asymmetrically distorted, as in the saddle-shaped heme observed in X-ray studies of cytcohrome c,24 it contains no mirror planes, and is therefore chiral. Solution circular dichroism spectra of cytochrome c have revealed a chiroptical response near 400 nm associated with electronic transitions (Soret band) in the heme group.25-27 Additionally, since the linear chiroptical response of the heme changes with oxidation state,25 SHG-CD may be sensitive to the redox state of the adsorbed protein via the heme chirality. SERRS has been shown to be a powerful in situ probe of hemoprotein oxidation states and structures at metal surfaces.28-30 Since the chirality and oxidation state of the heme in cytochrome c are intrinsically linked, it is hoped that a comparison of both SHG-CD and SERRS measurements will allow for a more complete description of cytochrome c at membrane surfaces. Materials and Methods31 Horse heart cytochrome c was obtained from Sigma Chemical Co. (purity >98%) and used without further purification. Cytochrome c solutions were prepared in HPLC grade water (filtered through 0.1 µm filters) obtained from Fisher Scientific Co. Phosphate buffers (4 mM, pH 7)32 were made using dihydrogen potassium phosphate (Aldrich Chemical Co.), degassed, and filtered before use. Reduced cytochrome c was prepared by adding excess ascorbate (>10 molar excess) adjusted to the same pH as the protein solution. Surface-adsorbed cytochrome c was exposed to reducing conditions by adding excess ascorbate, at the same pH, to the solution in contact with the surface. Measurements at the fused silica/water interface were performed using the flat face of a hemispherical lens (Virgo Optics). The initial substrates for the SAM and HBM surfaces were fused silica windows (Esco Optics Co., flatness λ/4). The hemispherical lens, the fused silica substrates, and all the glassware used in our experiments were cleaned in a sulfuric acid/NoChromix (Godax Laboratories) mixture and rinsed in high-purity water until neutral pH was reached. (23) Byers, J. D.; Hicks, J. M. Chem. Phys. Lett. 1994, 231, 216-224. (24) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990, 214, 585-595. (25) Myer, Y. P. In Methods in Enzymology; Fleischer, S., Packer, L., Eds.; Academic Press: New York, 1978; Vol. 54, pp 249-284. (26) Woody, R. W. In Circular Dichroism: Principles and Applications; Nakanishi, K., Berova, N., Woody, R. W., Eds.; VCH: New York, 1996; pp 489-501. (27) Blauer, G.; Sreerama, N.; Woody, R. W. Biochemistry 1993, 32, 6674-6679. (28) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 60106021. (29) Hildebrandt, P. In Cytochrome c: A Multidisciplinary Approach; Scott, R. A., Ed.; University Science Books: Sausalito, CA, 1996; pp 303-325. (30) Cotton, T. M.; Kim, J.-H.; Chumanov, G. D. J. Raman Spectrosc. 1991, 22, 729-742. (31) Certain commercial products are identified to adequately specify the experimental procedures; this does not imply endorsement or recommendation by NIST. (32) The accepted SI unit of concentration, mol/L, has been represented by the symbol M to conform to the conventions of this journal.
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The alkanethiol monolayers and HBMs were selfassembled at gold-coated fused silica surfaces. Sputtered 500 Å thick gold films with a 4 Å chrominium adhesion layer were found to be stable in contact with the aqueous solutions and at the laser powers in our experiments. The gold-coated fused silica was transferred from the vacuum chamber immediately into a 1 mM thiol solution and allowed to incubate overnight. For the methyl-terminated SAMs, dodecanethiol (Fluke, purity >98%) was used. The carboxylic acid-terminated SAMs were made using 16mercaptohexadecanoic acid (synthesized by D. Vanderah, NIST). Upon removal from the thiol solution, the SAMs were rinsed and sonicated in ethanol. The HBMs were prepared by adding a monolayer of lipid to a methyl-terminated SAM; phospholipid vesicles were added to the buffer solution above the dodecanethiol SAM in the sample cell, and the spontaneous addition of a single monolayer of phospholipid to the alkanethiol SAM surface was followed in parallel experiments with both surface plasmon resonance (SPR) and SHG measurements. Unless otherwise noted, vesicles were added in 100 mM NaCl in 4.4 mM phosphate buffer, pH 7.0 (phosphate-buffered saline (PBS)). Since at physiological pH cytochrome c binds strongly to mixed anionic and zwitterionic phospholipid vesicles,1 the HBMs were made from phospholipid vesicles containing 20 mol % dioleoylphosphatidic acid (DOPA), an anionic phospholipid, and 80 mol % 1-palmitoyl-2-oleoylphosphatidyl choline (POPC) or dimyristoylphosphatidyl choline (DMPC), zwitterionic phospholipids. All experiments were performed at room temperature. Verification that the cytochrome c solutions were reducible in solution by ascorbate was obtained via CD spectroscopy using a Jasco 710 spectropolarimeter. CD spectra were background-corrected and smoothed using a Savitzsky-Golay approach. The oxidized cytochrome c sample was 30 µM in 4.4 mM phosphate buffer, pH 7.0. The reduced cytochrome c sample was obtained by adding a 2 molar excess of ascorbate in pH 7.0 buffer to the oxidized cytochrome c sample. Surface plasmon resonance measurements were performed to independently verify the formation of the HBM and the adsorption of cytochrome c to the SAM and HBM surfaces. SPR has been used previously to monitor the formation of HBMs and the interactions of proteins with SAMs and HBMs.14,33 The SPR instrument used in this study was custom-built and has been described in detail elsewhere.33 SPR measurements were performed at a fixed wavelength of monochromatic light which was focused onto the sample to probe a range of incident angles. The angle at which the incident beam couples maximally into surface plasmons (indicated by minimum reflected intensity) is a function of the film thickness and refractive indices of the interface. The entire cone of reflected light is imaged onto a charge-coupled device (CCD) detector, and from the intensity minimum, the angle of maximum coupling of radiation into the plasmons is obtained as a location on the CCD detector (in pixel number). Data points were taken every 10 s and fit to a parabola to find the location of minimum reflected intensity. Changes in surface layer thickness or the bulk solution refractive index cause the resonant incident angle to move; the SPR data reported here have been corrected for bulk refractive index changes. Using the previously established relationships between refractive index and pixel position, the SPR data (33) Silin, V.; Weetall, H. H.; Vanderah, D. J. J. Colloid Interface Sci. 1997, 185, 94-103.
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may be expressed as a surface thickness as a function of time.33 All SPR measurements were done under conditions of constant flow at 0.25 mL/min, with a cell volume of 30 µL. SERRS provided evidence regarding the conformation and redox activity of the HBM-adsorbed cytochrome. Details of the SERRS experimental system have been published previously.34 For the SERRS experiments, the HBMs were formed on 3 mm diameter Ag (Aldrich 99.9%) rods encased in Teflon sleeves that left the surface open to solution. The metal was electrochemically roughened, rinsed with water and ethanol, and then placed in 1 mM ethanolic solutions of hexanethiol to form an alkanethiol monolayer. The lipid layer of the HBM was added via fusion of phospholipid vesicles composed of 20 mol % DOPA and 80 mol % DMPC in PBS. Both phospholipid components were fully deuterated. After 30 min of incubation, the cell was rinsed with PBS and the solution replaced with a low ionic strength buffer (LIB) of 4.4 mM potassium phosphate, pH 7.0 (ionic strength 10 mM). Cytochrome c was added as a 30 µM solution in the low ionic strength buffer. The cell was rinsed and refilled with the same buffer after 30 min to remove the cytochrome c in solution. All electrochemical potentials were measured with respect to a Ag/AgCl reference electrode and were converted to values versus the normal hydrogen electrode to facilitate comparison with pertinent literature. SERRS spectra were collected using a SPEX 1404 double stage monochromator (slit width 500 µm) converted to single-stage use and connected to a SPEX Spectrum CCD. An argon ion laser provided 514.5 nm excitation light which is resonant with electronic transitions of the cytochrome c heme. The angle of incidence on the electrode was ca. 60°. Scattered radiation was collected at 90° by a 50 mm focal length lens, passed through a holographic laser-line filter, and focused on the entrance slit of the monochromator. Spectra were obtained by averaging 50300 1 second scans. Data acquistion was performed using SPEX DM3000 software. Data analysis was performed using GRAMS 386 software (Galactic Industries). The laser system used for the SHG and SHG-CD studies consisted of a mode locked titanium-sapphire (Ti-S) laser (built after the Washington State design35) pumped by a beam-locked argon ion laser (Spectra Physics 2060). The Ti-S laser pulses were 150 fs full width at half-maximum (fwhm) with energies of approximately 5 nJ/pulse. The sample cell consisted of a glass dome (volume 5 mL) filled with aqueous solution in contact with an HBM or SAM. The polarization of the fundamental laser light was controlled using a half waveplate (for linearly polarized light) or quarter waveplate (for circularly polarized light). The incidence angle used was in the range of 45-51° from the surface normal. The laser light was focused at the interface using a 10 cm focal length lens and reflected from the sample surface along with the second harmonic signal. The fundamental light was blocked with a dichroic filter, and the SHG was detected using a monochromator and a low dark count photomultiplier tube (Hamamatsu R585). A two-channel gated photon counter (Stanford Research SR445) was used for the data collection after amplification of the signal (Stanford Research SR440). A reference SHG signal was (34) Meuse, C. W.; Niaura, G.; Lewis, M. L.; Plant, A. L. Langmuir 1998, 14, 1604-1611. (35) Murnane, M.; Kapteyn, H. C.; Huang, C.-P.; Asaki, M. T.; Garvey, D. Mode-locked Ti:Sapphire Laser; Washington State University: Pullman, WA, 1992.
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Figure 1. Schematic showing surface second harmonic generation whereby light at frequency ω is converted to light at 2ω. The plane of incidence is the plane defined by the incident and reflected beam. Light polarized in the plane of incidence is designated p-polarized light. Light that is polarized in the plane parallel to the surface is designated s-polarized light.
Figure 2. Soret CD spectra of 30 µM oxidized (s) and reduced (‚‚‚) cyt cochrome c in 4.4 mM phosphate buffer (pH 7.0) in a 1 mm path length cell.
generated in transmission simultaneously through a monopotassium phosphate suspension (particle size
