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Protein Deformation of Lipid Hybrid Bilayer Membranes Studied by Sum Frequency Generation Vibrational Spectroscopy Andrew W. Doyle,†,| Joerg Fick,‡ Michael Himmelhaus,‡ Wolfgang Eck,‡,| Irene Graziani,§ Igor Prudovsky,§,| Michael Grunze,‡,| Thomas Maciag,§,| and David J. Neivandt*,†,| Department of Chemical and Biological Engineering, University of Maine, Orono, Maine 04469, Department of Applied Physical Chemistry, University of Heidelberg, D-69120 Heidelberg, Germany, Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04704, and Institute for Molecular Biophysics, The Jackson Laboratory, Bar Harbor, Maine 04609 Received June 25, 2004. In Final Form: August 13, 2004 Structural deformations of lipid hybrid bilayer membranes induced by signal peptideless (SPL) proteins have been studied for the first time using the inherently surface specific nonlinear optical technique of sum frequency generation vibrational spectroscopy. Specifically, deformations of 1,2-distearoylphosphatidylglycerol (DSPG) membranes induced by interaction with FGF-1, a SPL protein which is released as a function of cellular stress through a nonclassical pathway, have been investigated. FGF-1 was found to induce lipid alkyl chain deformations in previously highly ordered DSPG membranes at the extremely low concentration of 1 nM at 60 °C. The deformation process was shown to exhibit a degree of reversibility upon removal of the protein by rinsing with buffer solution.
Introduction A growing number of secreted proteins have been demonstrated to be devoid of classical signal sequences in their primary structures and thus are unable to utilize the endoplasmic reticulum (ER)-Golgi pathway for their release. The signal peptideless (SPL) proteins however play a significant role in the regulation of a variety of critical cellular activities such as growth, differentiation, and migration.1-4 The nonclassical mechanism by which SPL polypeptides access the extracellular milieu is however not well understood. Elucidation of the transport mechanism may potentially result in the design of therapeutics targeted at regulating transport and hence the management of clinical disorders induced by SPL polypeptide transport. For example the fibroblast growth factor (FGF) gene family includes the prototype members FGF-1 and FGF-2 that lack signal peptides,2 are major regulators of embryogenesis, and play critical roles in angiogenesis, certain types of cancer and restenosis.1-3,5,6 In this work, FGF-1 interactions with hybrid bilayer membranes are characterized for the first time using sum frequency generation vibrational spectroscopy (SFS) in * To whom correspondence should be addressed: dneivandt@ umche.maine.edu; tel +1 207 581-2288; fax +1 207 581-2323. † Department of Chemical and Biological Engineering, University of Maine. ‡ Department of Applied Physical Chemistry, University of Heidelberg. § Center for Molecular Medicine, Maine Medical Center Research Institute. | Institute for Molecular Biophysics, The Jackson Laboratory. (1) Burgess, W. H.; Maciag, T. Annu. Rev. Biochem. 1989, 58, 575. (2) Friesel, R.; Maciag, T. Thromb. Haemostasis 1999, 82, 748. (3) Szebenyi, G.; Fallon, J. F. Int. Rev. Cytol. 1999, 185, 45. (4) Prudovsky, I.; Mandinova., A.; Soldi, R.; Bagala, C.; Graziani, I.; Landriscina, M.; Tarantini, F.; Duarte, M.; Bellum, S.; Doherty, H.; Maciag T. J. Cell Sci. 2003, 116, 4871. (5) Folkman, J. Semin. Oncol. 2002, 29, 15. (6) Mandinov, L.; Mandinova, A.; Kyurkchiev, S.; Kyurkchiev, D.; Kehayov, I.; Kolev, V.; Soldi, R.; Bagala, C.; De Muinck, E. D.; Lindner, V. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6700.
order to probe the membrane deformations associated with the nonclassical transport process. In response to cellular stress,4 such as heat shock, hypoxia, and serum starvation, FGF-1 is known to gain access to the extracellular compartment in the form of a Cu2+-mediated polypeptide complex that forms on the interior side of the plasma membrane.7,8 Specifically, FGF-1 is exported as a Cys30 homodimer noncovalently complexed with S100A13 and the p40 extravesicular domain of the Synaptotagmin (Syt)1.9-11 Each of the polypeptides involved in the release of FGF-1 has been demonstrated to have an affinity for both Cu2+ and acidic phospholipids (pLs), which are known to be capable of flipping from the inner leaflet of the plasma membrane to the outer leaflet in response to cellular stress, including heat shock.4 Preliminary and published data12 suggest that FGF-1, as well as the individual components that mediate its nonclassical release, is able to associate with acidic phospholipids and to permealize liposomes comprising such pLs in a temperature- and concentrationdependent manner. It may be hypothesized therefore that conformational changes of the lipid bilayer may be involved in facilitating the transport of FGF-1 through the cell membrane. To evaluate the role of the structure of the phospholipid component of the plasma membrane in the nonclassical transport process, the present work has employed the surface specific optical technique of SFS to probe FGF-1 induced deformations of acidic phospholipid model mem(7) Landriscina, M.; Bagala, C.; Mandinova, A.; Soldi, R.; Micucci, I.; Bellum, S.; Prudovsky, I.; Maciag, T. J. Biol. Chem. 2001, 276, 25549. (8) Prudovsky, I.; Bagala, C.; Tarantini, F.; Mandinova, A.; Soldi, R.; Bellum, S.; Maciag, T. J. Cell Biol. 2002, 158, 201. (9) Tarantini, F.; LaVallee, T.; Jackson, A.; Gamble, S.; Mouta Carreira, C.; Garfinkel, S.; Burgess, W. H.; Maciag, T. J. Biol. Chem. 1998, 273, 22209. (10) Jackson, A.; Tarantini, F.; Gamble, S.; Friedman, S.; Maciag, T. J. Biol. Chem. 1995, 270, 33. (11) Landriscina, M.; Soldi, R.; Bagala, C.; Micucci, I.; Bellum, S.; Tarantini, F.; Prudovsky, I.; Maciag, T. J. Biol. Chem. 2001, 276, 22544. (12) Mach H.; Middaugh, C. R. Biochemisty 1995, 34, 9913.
10.1021/la0484220 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/10/2004
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branes. Briefly, SFS provides vibrational spectra of molecules at interfaces.13,14 SFS relies on the nonlinear optical phenomenon of sum frequency generation (SFG). SFG occurs when two pulsed laser beams, one of fixed near-infrared or visible frequency and the other of tunable infrared frequency, achieve spatial and temporal overlap at an interface. Light is emitted at the sum of the two incident frequencies. The intensity of the light is resonantly enhanced when the frequency of the tunable infrared beam coincides with a vibrational mode of the molecules adsorbed at the interface. By detection of the sum frequency (SF) light as a function of infrared frequency, a vibrational spectrum is obtained which is upshifted into the visible region of the electromagnetic spectrum. The selection rules for SF activity differ from those for linear spectroscopy (such as those for infrared or Raman spectroscopies). Specifically, in order for a vibrational resonance to be SF active, it must be in an asymmetric, i.e., noncentrosymmetric, environment. The requirement for asymmetry may be satisfied on both a macroscopic and a molecular level. On a macroscopic scale, an isotropic distribution of molecules in a bulk phase (for example, lipids in an aqueous solution) is centrosymmetric, that is, lacks asymmetry and is consequently SF inactive. Introduction of an interface into an isotropic bulk phase, and the resultant production of a surface population of molecules otherwise isotropically distributed in solution, gives rise to a plane of asymmetry and the SF activity of exclusively the interfacial molecules. It is noted however that in order for the interfacial molecules to be SF active, they must have a net polar orientation. No SF emission arises from molecules arranged in an equal number of opposite orientations on a surface or from a completely disordered surface structure. SFS is consequently inherently interfacially specific and does not suffer from the difficulties associated with the signal from the surface being swamped by the signal from the bulk medium, as experienced by linear spectroscopies.15 On a molecular level, the asymmetry requirement for SF activity facilitates quantification of the degree of order of molecules susceptible to conformational change. For example each methylene group in a highly ordered, fully trans, hydrocarbon chain is in a locally centrosymmetric environment and hence SF inactive. The introduction of gauche defects into such hydrocarbon chains however breaks the centrosymmetry and results in SF activity of the affected methylene groups. In addition to obtaining conformational information of interfacial molecules, the phase of an SF signal also reports on the orientation of the species under investigation. Indeed whether vibrational resonances appear in spectra as “peaks” or “dips” provides an absolute measure of the polar orientation of the surface species. Surprisingly, the bulk of SF studies to date have focused on synthetic polymer and/or surfactant systems; very few attempts have been made to extend the technique to lipid systems of biological import. The Richmond SF group has published a considerable number of surfactant adsorption studies at the water/oil interface16,17 and has recently extended these studies to exploratory investigations of interfacial lipid conformations at comparable interfaces.18,19 Similarly SFS has been used in conjunction with (13) Bain, C. D.; J. Chem. Soc., Faraday Trans. 1995, 91, 1281. (14) Shen, Y. R. Nature 1989, 337, 519. (15) Adamson A. W.; Gast, A. P. Physical Chemistry of Surfaces; John Wiley & Sons: New York, 1999. (16) Watry M. R.; Richmond, G. L. J. Am. Chem. Soc. 2000, 122, 875. (17) Walker, R. A.; Conboy, J. C.; Richmond, G. L. Langmuir 1997, 13, 3070. (18) Smiley B. L.; Richmond, G. L. Biopolymers 2000, 57, 117.
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Fourier transform infrared spectroscopy to probe the fluidity of pLs cast onto solid substrates in relation to the order/disorder state of their hydrocarbon chains.20 The Bonn group has recently studied the phase diagram of phosphocholine at the air/water interface as a function of lateral pressure.21 Finally, the feasibility of studying vesicle fusion at the solid/liquid interface via SFS has been demonstrated by Petralli-Mallow et al.22 In the present study SFS has been employed to investigate the polar orientation and degree of alkyl chain conformational order of 1,2-phosphatidylglycerol (DSPG) hybrid bilayer membranes (HBMs) as a function of interaction with nanomolar concentrations of recombinant FGF-1. The findings have implications with regards to the role of the structure of acidic phospholipid components of plasma membranes in the nonclassical transport process of signal peptideless proteins such as FGF-1. Experimental Section Substrates employed consisted of optically polished silicon wafers coated with a primer layer of 5 nm of titanium followed by a 100 nm layer of gold. Both metals were thermally evaporated onto the substrate employing a home-built evaporator operated at a base pressure of 2 × 10-7 mbar. A self-assembled monolayer of perdeuterated octadecanethiol (d-ODT) was created on the gold-coated substrates employing the following procedure: The coated wafers were exposed to a 2 h UV cleaning treatment, incubated in a 10 µM solution of d-ODT in methanol overnight, rinsed twice, and sonicated in methanol for 5 min. Ellipsometric analysis of the substrates employing a J.A. Woollam M-44 ellipsometer and a three-layer refractive index model23 revealed a d-ODT film thickness of 24 ( 1 Å. Unilammelar vesicles were produced via an extrusion technique developed and recommended by Avanti Polar Lipids (APL), using an APL miniextruder.24 Twenty five micromole of DSPG was dissolved in 1 mL of chloroform employing a 5 mL glass vial. The chloroform was allowed to evaporate as the container was rotated resulting in the deposition of a thin lipid film on the inside vial wall. This facilitated the hydration of the lipid film by a 1 mL solution of 1× phosphate buffered saline (1× PBS ) 0.138 M NaCl; 0.0027 M KCl, pH 7.4) producing large multilammelar vesicles. The vesicle suspension was subsequently warmed to and maintained at 60 °C, above the phase transition temperature of DSPG (55 °C). The APL miniextruder equipped with a 0.1 µm membrane was employed to create nominally 100 nm unilamellar vesicles from the multilammelar vesicle solution via 10 passes through the membrane. The vesicle suspension was diluted to 0.5 mM DSPG immediately prior to use. SF spectra were recorded on a femtosecond broadband SF spectrometer similar to that presented by Ye and co-workers.25 Briefly, the output of a Ti:sapphire oscillator (Coherent Vitesse, USA) at 805 nm is amplified in a regenerative amplifier (Quantronix Titan, USA) to give 100 fs pulses at 3 mJ/pulse and a repetition rate of 1 kHz. Eighty percent of this fundamental is used to pump a TOPAS optical parametric generator and amplifier (Light Conversion, Lithuania) with subsequent noncollinear down-conversion (Light Conversion) generating ∼120 fs pulses in a spectral range from ∼4000 to ∼1000 cm-1. The remaining 20% of the pump beam is fed into a home-built spectral shaper, which forms narrow NIR pulses of ∼15 cm-1 in bandwidth at ∼805 nm. Typical pulse energies were 9 µJ for the narrowed (19) Walker, R. A.; Gragson, D. E.; Richmond, G. L. Colloid Surf., A 1999, 154, 175. (20) Lobau, J.; Sass, M.; Pohle, W.; Selle, C.; Koch, M. H. J.; Wolfrum, K. J. Mol. Struct. 1999, 481, 407. (21) Roke, S.; Schins, J.; Muller, M.; Bonn, M. Phys. Rev. Lett. 2003, 90, 128101. (22) Petralli-Mallow, T.; Briggman, K. A.; Richter, L. J.; Stephenson, J. C.; Plant, A. L. Proc. SPIE 1999, 25, 1. (23) Tokumitsu, S.; Liebich, A.; Herrwerth, S.; Eck, W.; Himmelhaus, M.; Grunze, M. Langmuir 2002, 18, 8862. (24) Nayar, R.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1989, 986, 200. (25) Ye, S.; Noda, H.; Morita, S.; Uosaki, K.; Osawa, M. Langmuir 2003, 19, 2238.
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Figure 1. In situ SF spectra recorded in the ppp beam polarization combination of the formation and FGF-1 protein deformation of 1,2-distearoylphosphatidylglycerol (DSPG) lipid HBMs at 60 °C. (a) After vesicle fusion of a 0.5 mM DSPG in 1× PBS unilamellar suspension with a perdeuterated octadecanethiol covered gold substrate for 90 min. (b) Having rinsed the HBM of (a) with a 1× PBS buffer solution for 30 min. (c) The effect of the addition of a solution of 1 nM FGF-1 in 1× PBS to the HBM of (b) for 30 min. (d) After rinsing the HBM of (c) with a 1× PBS buffer solution for 30 min. Spectra are presented on the same normalized intensity scale but are displaced vertically for clarity. near-infrared and ∼15 µJ for the mid-infrared beam around 2900 cm-1, i.e., in the CH stretching vibrational region, respectively. The beams were p-polarization selected, focused, and coupled into a flow cell through a CaF2 prism to yield a spot size of 1 mm on the sample’s surface. To minimize absorption of the midinfrared beam in the aqueous environment of the flow cell, the gap between prism and sample surface is set to a few micrometers during the measurements. The generated broadband SFG signal is analyzed by a high-resolution detection system consisting of a 300 mm monochromator (Acton Research, USA) and an imageintensified high-resolution CCD camera (Roper Scientific, USA). Typically, signals were collected for 10 min to obtain an acceptable signal-to-noise ratio. Spectra were baseline normalized via the fitting of Voigt profiles to the left and right wings, that is the regions from 3250 to 2980 cm-1 and below 2800 cm-1. Spectra were wavelength normalized to the methyl symmetric stretching mode of 2874 cm-1. 1,2-Distearoylphosphatidylglycerol (DSPG) was purchased from Avanti Polar Lipids (APL) Alabaster, AL, and was used as received. Perdeuterated octadecanthiol (d-ODT) was synthesized and purified by the Heidelberg group. Recombinant human FGF1 was prepared as described in ref 26. All other chemicals were obtained from Sigma-Aldrich and were used as received.
Results and Discussion Figure 1a presents the SF spectrum obtained after fusion of a DSPG unilamellar vesicle suspension with the d-ODT substrate in situ in a liquid flow cell27,28 at 60 °C. The temperature of 60 °C was chosen in order to replicate cellular heat shock and hence conditions known to lead to interaction of FGF-1 with phospholipid membranes.4 Specifically, the substrate was mounted in the flow cell and filled with a 1× PBS buffer solution introduced from an external reservoir via a peristaltic pump and capillary flow tubing. The cell temperature was regulated via resistive block heaters and an electronic feedback system. The solution reservoir was immersed in a thermostated (26) Engleka, K. A.; Maciag, T. J. Biol. Chem. 1992, 267, 11307. (27) Lambert, A. Ph.D. Thesis, University of Cambridge, 2001. (28) Windsor, R.; Neivandt, D. J.; Davies, P. B. Langmuir 2001, 17, 7306.
water bath also maintained at 60 ( 1 °C. The substrate was brought up to within micrometers of the CaF2 prism of the cell using a micrometer, and the spectrometer was aligned from maximum SF signal production and detection. A reference SF spectrum (not shown) was obtained, no resonances in the CH stretching region were detected indicating that the substrate was free of contaminants. The substrate was subsequently retracted from the prism into the body of the cell and the PBS solution replaced with a 0.5 mM unilamellar DSPG vesicle solution, without drying of the substrate. The vesicle solution was allowed to equilibrate for 90 min with the substrate under continuously flowing conditions. The flow was then stopped, the substrate brought back to within micrometers of the cell prism, and the SF spectrum presented in Figure 1a recorded. Investigation of Figure 1a reveals three strong SF resonances of positive phase at 2881, 2943, and 2973 cm-1. These resonances concur with those typically observed for alkyl chains in aqueous environments29,30 and are attributed to resonances of the terminal methyl groups of the lipid alkyl chains, specifically the CH3 symmetric stretching mode, a Fermi resonance of the symmetric stretching mode, and the in-plane CH3 asymmetric stretching mode, respectively. Spectral fitting and deconvolution (see Supporting Information for the details of the fitting procedure, the spectrum, and the resultant fitting parameters) reveals the presence of a weak outof-plane asymmetric methyl stretching mode and weak methylene resonances (specifically the methylene symmetric stretching mode at approximately 2860 cm-1 and a broad resonance, treated as an integrated mode for the purpose of fitting, attributed to a combination of a weak asymmetric methylene stretching mode and a Fermi resonance of the methylene symmetric stretching mode in the region 2890-2930 cm-1 29,31). These findings are indicative of a very highly ordered lipid layer on the d-ODT substrate. Indeed the presence of only weak methylene resonances in the SF spectrum implies that the alkyl chains of the lipid are in a locally near centrosymmetric, and therefore only weakly SF active, predominantly alltrans environment. The occurrence of the methyl resonances as spectral peaks (that is with positive phase) implies that the polar orientation of the lipid is with their headgroups into solution and their alkyl chains toward the hydrophobic substrate, consistent with expectations. This conclusion derives from the use of a gold substrate and the consequent dependence of the phase of the spectral resonances upon a cross term of the nonresonant signal arising from the gold and the resonant terms arising from the lipid. The observation of a highly ordered lipid layer after introduction of a vesicle solution to a hydrophobic substrate is consistent with the phenomenon of vesicle fusion. Indeed the SF spectrum of Figure 1a is comparable to that obtained by Petralli-Mallow et al. in their study demonstrating the feasibility of application of SFS to the investigation of vesicle fusion employing DMPC.22 It was noted by Petralli-Mallow et al. that rinsing the adsorbed lipid layer with a buffer solution after the equilibration procedure led to a significant strengthening of the methyl resonances. This finding was attributed to the removal from the substrate of unfused vesicles and/or lipid multilayers without disruption of the underlying ordered (29) Ward, R. N. Ph.D. Thesis, University of Cambridge, 1993. (30) Casford, M. T. L.; Davies, P. B.; Neivandt, D. J. Langmuir 2003, 19, 7391. (31) Macphail, R. A.; Snyder, R. G.; Strauss, H. L. J. Chem. Phys. 1982, 77, 1118.
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lipid monolayer, consistent with the HBM formation mechanism proposed by Plant and co-workers based on surface plasmon resonance studies.32 Consequently in the present work a 30 min rinse of the substrate with PBS buffer was performed by retraction of the sample from the prism and replacement of the vesicle solution with 1× PBS. The substrate was subsequently brought back up to the prism and the spectrum of Figure 1b recorded. In common with the findings of Petralli-Mallow et al. the intensity of each of the methyl resonances in the SF spectrum is observed to greatly increase with respect to the nonrinsed spectrum (Figure 1a). Spectral fitting and deconvolution (see Supporting Information) reveal that in addition to the methyl resonance intensity increases, a significant decrease in the methylene resonance intensities is detected. This finding is consistent with the concept of the removal of disordered lipid from the interface as a result of the rinsing procedure leaving a hybrid bilayer membrane of highly ordered DSPG with alkyl chains in predominantly trans conformers adsorbed on the d-ODT layer with their headgroups oriented into solution. The effect of the addition of the signal peptideless protein FGF-1 on the conformational order of the HBM was determined by retracting the substrate from the prism, replacing the PBS buffer solution with a 1 nM solution of FGF-1 in 1x PBS, equilibrating the system for 30 min and recording an SF spectrum with the substrate in the approach position. The resulting spectrum is presented in Figure 1c. Investigation of Figure 1c reveals that the intensity of all of the methyl modes of DSPG have been significantly decreased with reference to the unperturbed membrane, Figure 1b, as a result of interaction of FGF-1. This finding indicates that the methyl groups of DSPG have experienced a loss of symmetry, that is been forced into a more isotropic, disordered conformation as a result of perturbation of the membrane by the protein. Coincident with the loss of methyl group SF intensity is the strengthening in the spectra of resonances attributable to the methylene groups of the alkyl chains of the phospholipid. Specifically, the resonance at approximately 2860 cm-1 arising from the methylene symmetric stretching mode appearing in the spectrum of Figure 1c as a shoulder on the low wavenumber side of the methyl symmetric stretching mode, and the broad resonance attributed to a combination of a weak asymmetric methylene stretching mode and a Fermi resonance of the methylene symmetric stretching mode in the region 28902930 cm-1. Figure 2 presents the results of spectral fitting and deconvolution of Figure 1c. The presence of significant methylene resonances is clearly evident in Figure 2. The resultant fitting parameters (see Supporting Information) confirm the coincident decrease in methyl mode intensities and increase in methylene mode intensities. The strengthening of methylene mode intensities in the SF spectrum of the protein-modified HBM is indicative of an increase in the number of gauche defects in the alkyl chains of DSPG, further breaking of methylene centrosymmetry present in the previously predominantly trans conformers, and hence increased SF activity. The observation of a loss of methyl group anisotropy, coupled with the introduction of gauche defects in the phospholipid methylene chain, results in the conclusion that that FGF-1 induces conformational deformations of DSPG hybrid bilayer membranes at the extremely low but biologically relevant concentration of 1 nM at a temperature of 60 °C. This finding is consistent with work by the authors and co(32) Hubbard, J. B.; Silin, V.; Plant, A. L. Biophys. Chem. 1998, 75, 163.
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Figure 2. Upper: The SF spectrum of Figure 1c (open circles) and the spectral fit (solid line) obtained via a least-squares fitting procedure as described in the Supporting Information. Lower: Intensities, |χq(2)|2, of the individual Lorentzian profiles as obtained from the least squares spectral fitting procedure. Negative profiles indicate an orientation of the transition dipole moment of the respective resonance in the +z direction, that is, away from the gold substrate.
workers demonstrating the ability of FGF-1 to permealize phosphatidylglycerol vesicles at significantly higher concentration (62.5 nM) and lower temperature (50 °C) conditions.12 As such the present work demonstrates the extreme sensitivity of SFS to the detection of membrane deformation processes induced by proteins involved in nonclassical transport. Further, it is concluded that FGF-1 disrupts, rather than induces order in, acidic phospholipid HBM structures under conditions replicating cellular heat shock. Finally, the effect on the conformational order of the FGF-1 deformed HBM of rinsing with PBS to remove the protein was determined. Specifically, the substrate was retracted from the prism, the vesicle solution was replaced with a 1× PBS solution which was flown for 30 min prior to the recording of an SF spectrum in the approach position (Figure 1d). Comparison of the spectrum of Figure 1d with that of Figure 1b (spectrum of the rinsed HBM prior to addition of FGF-1) reveals that they are qualitatively comparable. Spectral fitting and deconvolution of Figure 1d (see Supporting Information) reveal that the decrease in methyl resonance intensities and hence conformational order induced by the addition of FGF-1 in Figure 1c has been reversed. Further, the methylene resonances present in Figure 1c and attributed to FGF-1 induced gauche deformations of the DSPG alkyl chains are less intense in Figure 1d and indeed have returned to approximately their pre-deformed values (see the deconvoluted spectrum and fitting parameters in the Supporting Information). This finding is indicative of the return of the phospholipid alkyl chain conformation to a more fully trans state. It is
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concluded that the deformation of a HBM membrane induced by FGF-1 displays a degree of reversibility, at least under the experimental conditions employed in the present work. Conclusion Hybrid bilayer membranes (HBMs) consisting of a perdeuterated octadecanethiol on gold lower layer and an acidic phospholipid, 1,2-distearoylphosphatidylglycerol, outer layer have been created via the technique of vesicle fusion at 60 °C. The membranes have been characterized in situ via the nonlinear optical technique of sum frequency generation vibrational spectroscopy and have been shown to be highly conformationally ordered with the lipid orientated with its headgroup into solution. Introduction of the signal peptideless protein FGF-1 at 1 nM concentration results in deformation of the HBM, observed as a loss of DSPG methyl group anisotropy and an in increase in the number of gauche defects in the alkyl chains of the lipid. These findings correlate with the known ability of FGF-1 to permealize phosphatidylglycerol liposomes. Rinsing the HBM to remove the protein led to the regaining
of a highly ordered DSPG conformation, suggesting that the process of deformation of the HBM by FGF-1 displays a degree of reversibility. Acknowledgment. Special thanks are extended to the Paul B. Davies SFS group, Department of Chemistry, Cambridge University, for the use of the liquid flow cell and to Alex G. Lambert for his drawing of the cell used in the TOC graphic. A.W.D. gratefully acknowledges the Institute for Molecular Biophysics and the University of Maine Pulp and Paper Foundation for financial support. This work was supported by NSF EPSCoR Grant EPS0132384, NIH Grants RR15555 to T.M. and HL35627 and HL32348 to T.M. and I.P., and in part by ONR Grant N00014-98-1-0500, DFG Grant HI 693/2-1, and the European FP6-2002-NMP-1 Grant “Nanocues”. Supporting Information Available: Deconvolutions of the spectra of Figure 1, along with tabulated values of the resultant fitting parameters. This material is available free of charge via the Internet at http://pubs.acs.org. LA0484220
