J. Phys. Chem. B 2008, 112, 2281-2290
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Quantifying the Ordering of Adsorbed Proteins In Situ Jie Wang, Sang-Ho Lee, and Zhan Chen* Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed: September 19, 2007; In Final Form: NoVember 11, 2007
We have investigated the orientation and conformation of protein molecules at the polystyrene (PS)/protein solution interface using sum frequency generation (SFG) vibrational spectroscopy, supplemented by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). In this research, we studied fibrinogen as a model protein. SFG studies indicate that fibrinogen adopts a bent structure after adsorbing to the PS surface. A broad orientation distribution of fibrinogen coiled-coils at the interface has been quantified by combining SFG and ATR-FTIR measurements. Error analysis for such a deduced distribution was carried out. This research demonstrates that quantitative structural information such as orientational and conformational ordering of proteins at interfaces can be studied using SFG supplemented by other spectroscopic techniques.
1. Introduction Understanding protein and peptide structures at interfaces is crucial for the design and development of biomedical materials,1-3 marine anti-biofouling coatings,4 antimicrobial peptides overcoming bacterial drug resistance,5 and biosensors using enzymes and antibodies.6 Recently, sum frequency generation (SFG) vibrational spectroscopy has been applied to investigate structural information of proteins and peptides at various interfaces.7-29 Initial SFG studies on proteins and peptides were mainly focused on the spectra in the C-H stretching range, which provided structural information about protein hydrophobic side chains. To deduce more abundant conformational information of interfacial proteins and peptides, it is necessary to examine their SFG amide signals. We have demonstrated the feasibility of detecting SFG signals from protein amide I groups and differentiating various secondary structures of proteins/peptides such as R-helices, β-sheets, and turns from such SFG amide I signals at interfaces, showing that SFG can characterize protein/ peptide secondary structures at interfaces.14,15,17,26-29 It is necessary to develop data analysis methodology to quantitatively characterize the structural information of various secondary structural motifs of proteins and peptides at interfaces using SFG. Knoesen and colleagues studied aligned helical polypeptides on a surface using SFG.16 The Simpson group developed a theoretical approach and novel computer software to calculate both the vibrational and electronic second-order nonlinear transition polarizabilities of various protein nonlinear optical signals, including amide I signals for known protein structures.30 Our group recently applied group theory and a projection operator method to analyze hyper-Raman, SFG, and four-wave mixing spectra,31 developing a method to quantitatively determine the orientation of R-helical structures at interfaces using SFG polarization analysis. We also quantitatively deduced two distinct orientations of R-helical melittin in a single lipid bilayer,26 showed quantitative ways to determine antiparallel β-sheet orientation,15 and observed strong SFG chiral spectra from β-sheet structures at the interface, which can increase the number of measurable parameters for protein * To whom all correspondence should be addressed. E-mail:
[email protected]. Fax: 734-647-4865.
structural determination using SFG.15 Here, quantitative data analysis methods will be applied to characterize structures of proteins at interfaces, using fibrinogen as a model. Fibrinogen (∼340 kD) is a large protein involved in thrombosis. The native structure of fibrinogen has been described as trinodular, with three hydrophobic domains connected by R-helical coiled-coils (Figure 1a, stars indicate the binding sites for platelets. Figure 1b is a simplified presentation for a native fibrinogen molecule).10,32-36 Fibrinogen molecules are elongated 45 nm structures consisting of two outer D domains, each connected by a coiled-coiled segment to a central E domain. The E domain contains a nexus of chains that bond the two almost identical halves of a molecule together in a small globular region. The conformation of surface-bound fibrinogen has been shown to play an important role in thrombus formation. For example, after adsorption to a surface, platelet binding sites may be shielded, reducing the ability to form a thrombus. Therefore, it is important to deduce the conformation of fibrinogen at interfaces to understand surface biocompatibility and blood coagulation mechanisms. Previously, the adsorption behavior of fibrinogen to two biomedical polyurethanes and a perfluorinated polymer has been investigated.27 Changes in the secondary structure of adsorbed fibrinogen were monitored using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and SFG. SFG measurements were performed in the amide I range as well as in the C-H/N-H stretching range. Amide I signals from SFG demonstrate that fibrinogen has post-adsorption conformational changes that are dependent upon the polymer surface properties. For example, strong attenuation of the amide I and N-H stretching signals with increasing residence time was observed for fibrinogen adsorbed to poly(ether urethane), but not for the other two polymers. This change is not readily observed by ATR-FTIR. Differences in the observed spectral changes for fibrinogen adsorbed to each polymer are explained by different initial binding mechanisms and post-adsorption conformational changes. Structural changes of fibrinogen after adsorption to polystyrene (PS) were also examined at the PS/protein solution interface in situ in our group.28 Different behaviors of hydrophobic side chains and secondary structures of adsorbed fibrinogen mol-
10.1021/jp077556u CCC: $40.75 © 2008 American Chemical Society Published on Web 01/25/2008
2282 J. Phys. Chem. B, Vol. 112, No. 7, 2008
Figure 1. (a) Structure of a fibrinogen molecule (stars indicate the binding sites for platelets). (b) Simplified presentation of a fibrinogen molecule. (c,d) Possible conformations and orientations of adsorbed fibrinogen molecules on the PS surface.
ecules have been observed. Our results indicate that, upon adsorption, the hydrophobic PS surface induces fast structural changes of fibrinogen molecules by aligning some hydrophobic side chains in fibrinogen so that they face to the surface. Such structural changes of fibrinogen hydrophobic side chains are local changes, and do not immediately induce significant changes of the protein secondary structures. Our research also shows that the interactions between adsorbed fibrinogen and the PS surface can induce significant changes of protein secondary structures or global conformations, which occur on a much longer time scale. Protein molecules adsorbed on a surface may not have a single conformation or orientation, but a broad orientation/conformation distribution instead. Therefore, to understand proteinsurface interactions in detail and obtain quantitative knowledge of interfacial protein ordering, it is necessary to characterize such a distribution. In this paper, we will quantitatively examine the molecular conformation of protein, using fibrinogen as a model, adsorbed at the PS/protein solution interface in situ using SFG. This current research is based on the SFG amide I signals contributed from fibrinogen R-helical coiled-coils, and will focus on the determination of orientation distribution of such fibrinogen coiled-coils at the interface. It is well-known that the peak centers of amide I signals can be used to identify various secondary structural motifs.37-41 However, random coiled structures may also contribute amide I signals in the characteristic frequency range of the R-helical structure. This research shows that we can distinguish these two structures by analyzing SFG spectra collected using different polarization combinations or different input beam angles. Our data analysis indicates that adsorbed fibrinogen molecules initially have an average bent conformation at the PS/solution interface. The orientation distribution of coiled-coils can be determined by intensity ratios of SFG spectra collected using different polarization combinations and absolute intensity measurements. Such deduced results are further validated by polarized ATR-FTIR measurements. This research also confirms that more information regarding protein orientational and conformational ordering at the interface can be obtained by combining different spectroscopic techniques (e.g., SFG and ATR-FTIR) in the investigation. Our previous results on melittin orientations in a lipid bilayer show that two different orientations coexist for melittins, and the distributions for both orientations are quite narrow.26 Differently, here we found that, at the polymer/solution interface, the orientation distribution of adsorbed fibrinogen is quite broad. 2. Experimental Section 2.1. Samples. Bovine fibrinogen was purchased from Sigma and was used as received. Fibrinogen solution was made by dissolving fibrinogen in phosphate buffered solution (PBS) with a total ionic strength of ∼0.14 M and a pH value of 7.4. The PBS was made by using deionized water (18.2 MΩ cm)
Wang et al. obtained from a Millipore ultrapure water system. The concentration of the protein solution used in this experiment was 1 mg/mL. PS was purchased from Scientific Polymer Products, Inc. and used as received. PS films were made by spin coating a 2% solution (wt/wt in toluene) onto CaF2 prism substrates (purchased from ESCO Products) at 2500 rpm. The CaF2 prisms were cleaned in toluene and rinsed thoroughly with solvent before spin coating to ensure that the polymer surface was free of contamination. 2.2. SFG. SFG is a second-order nonlinear optical spectroscopic technique that has superb surface sensitivity.42-59 Details regarding SFG theories and measurements have been extensively published42-59 and will not be repeated here. The SFG experimental setup was similar to that described in our earlier publications and will not be described.59 A near total reflection experimental geometry was adopted to collect the SFG signal from interfacial fibrinogen in this work.14 2.3. Polarized ATR-FTIR. The ATR-FTIR technique is a well-established method to analyze protein secondary structures on surfaces or at interfaces.40 It can provide information regarding the orientation of such secondary structures. Our ATRFTIR system is a Nicolet 550 FTIR spectrometer, with a standard 45° ZnSe ATR cell. A ZnSe grating polarizer (from Optometrics LLC) was used to collect polarized ATR-FTIR spectra. The ZnSe cell was cleaned by toluene, and the thin polymer layers were made by solvent casting on the ZnSe crystal. In the experiments, a PBS solution was brought into contact with the polymer film first. After at least 2 h to allow equilibration, a background spectrum of the polymer film/buffer was recorded. A new spectrum was then recorded after replacing the PBS solution with a protein solution. The interfacial protein spectrum was obtained by subtracting the background spectrum from the new spectrum. In order to evaluate the contributions from bulk proteins in the solution, the protein solution was then changed to a PBS solution, and an ATR-FTIR spectrum was collected for comparison. 3. Data Analysis Regarding the SFG and ATR-FTIR Signals from r-Helical and Random Coil Structures In the SFG literature, some functional groups, such as methyl and phenyl groups, at interfaces have been extensively studied, and parameters characteristic of their vibrational modes are wellknown.60-64 But so far, only limited work has been published on quantitative SFG studies on R-helices in the amide I frequency range. We have studied both molecular hyperpolarizability and SFG susceptibility tensors for an R-helix, which is one of the most common secondary structural motifs in proteins and peptides, using group theory and projection operator.31 In the Supporting Information, the details to deduce R-helix hyperpolarizability are also listed. In this paper, the orientation as well as the orientational distribution information of R-helices at interfaces will be examined, using fibrinogen as a model. We believe that such research will facilitate structural studies of peptides and proteins at interfaces using SFG. 3.1. Nonlinear Optical Susceptibility of r-Helices at the Interface. We want to study the structural information of adsorbed proteins at an interface, e.g., at a solid/liquid interface. We showed in our previous publication that we should be able to treat such an interface as a thin layer of protein molecules.57 The molecular orientation information can be obtained by relating SFG susceptibility tensor elements χijk (i, j, k ) x, y, z) to the SFG molecular hyperpolarizability tensor elements βlmn (l, m, n ) a, b, c). The relationship between the laboratoryfixed axis system (x, y, z) and the molecule-fixed coordinate
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Figure 2. Calculated susceptibility as a function of orientation angle θ for δ distribution: (a) χyyz of R-helix; (b) χyzy of R-helix; (c) χzzz of R-helix. (d) Susceptibility tensor elements of “free CdO” group.
system (a, b, c) can be specified by the Euler angles (φ, θ, ψ). The c-axis is parallel to the principal axis of the R-helix, and θ is the angle between the z-axis in the lab frame (here it is perpendicular to the surface) and the c-axis. φ is the azimuthal angle. As we showed previously,31 assuming the adsorbed protein molecules at the interface have azimuthal symmetry, we can deduce the susceptibility tensor elements for R-helices: For the A mode:
1 χA,xxz ) χA,yyz ) Ns[(1 + r)〈cos θ〉 - (1 - r)〈cos3 θ〉]βccc 2 1 χA,xzx ) χA,yzy ) χA,zxx ) χA,zyy ) Ns[(1 - r)(〈cos θ〉 2 〈cos3θ〉)]βccc χA,zzz ) Ns[r〈cos θ〉 + (1 - r)〈cos3 θ〉]βccc
(1)
where r ) βaac/βccc. For the E1 mode:
3.2. Nonlinear Optical Susceptibility of Random Coiled Structure at the Interface. Usually, amide I signals contributed from different secondary motifs in a protein can be separated in the frequency domain using vibrational spectroscopy. For example, an R-helix has its characteristic amide I frequency in the range of 1650-1660 cm-1. However, sometimes random coil structures may also contribute amide I signals in this spectral range. Therefore, assignment of the amide I signals between 1650 and 1660 cm-1 to an R-helix needs to be carefully examined.68 For SFG, because of the “random” or “disordered” character of some of these random coil structures, they may not contribute SFG signals under the electric dipole approximation. On the other hand, some such groups may form some more or less ordered structures and thus generate SFG signals. In the following, we will use “free CdO groups” to represent these structures. For “free CdO groups”, we have
1 χA,xxz ) χA,yyz ) Ns[(1 + r)〈cos θ〉 - (1 - r)〈cos3 θ〉]βccc 2
χE,xxz ) χE,yyz ) -Ns(〈cos θ〉 - 〈cos3 θ〉)βaca
1 χA,xzx ) χA,yzy ) χA,zxx ) χA,zyy ) Ns[(1 - r)(〈cos θ〉 2 〈cos3 θ〉)]βccc
χE,xzx ) χE,yzy ) χE,zxx ) χE,zyy ) Ns(〈cos3 θ〉βaca
χA,zzz ) Ns[r〈cos θ〉 + (1 - r)〈cos3 θ〉]βccc
χE,zzz ) 2Ns(〈cos θ〉 - 〈cos3 θ〉)βaca
(2)
Here Ns is the surface density of R-helical repeat units. Since the SFG hyperpolarizability can be deduced from the Raman and IR properties of the R-helical molecule, we deduced the relations among different hyperpolarizability tensor elements to be r ≈ 0.54 and βaca ≈ 0.32βccc from the experimental data of polarized Raman and IR.41,65-67
(3)
where r ) βaac/βccc ≈ 0.13 from ref 69. 3.3. Numerical Presentations. The relations between SFG susceptibility tensor elements and the orientation angle for R-helices and “free CdO groups” have been shown in the above section. Such relations can be plotted graphically. It depicts such relations assuming a δ distribution for the orientation angles. Figure 2 shows clearly that the relative intensity of the A and E1 modes of an R-helix usually vary greatly in SFG spectra collected by different polarization combinations of the input and
2284 J. Phys. Chem. B, Vol. 112, No. 7, 2008 output laser beams. In certain orientation angle ranges of some spectra, the SFG signal from the A mode is dominant (e.g., for small angles in χyyz). In other polarization combinations, contributions from the E1 mode can be dominant (e.g., for small angles in χyzy). The typical frequency difference between the A and E1 mode is about 5 cm-1;41 therefore we should be able to observe these frequency differences by collecting SFG spectra with different polarization combinations. We should point out that the “free CdO” groups in different local environments may have varied orientations and generate different peak centers. They may contribute SFG signals that can also behave differently under different polarization combinations. However, it is unlikely that the SFG signals generated from “free CdO” groups would be similar to those from R-helices in spectra collected from all polarization combinations. This polarization analysis can be used to distinguish SFG signals contributed by R-helices from those by “free CdO” groups, even though they appear to be in a similar frequency range. We should emphasize here that a δ distribution has been assumed when generating Figure 2. The above analysis to distinguish R-helices and “free CdO groups” for a very broad distribution should be similar to that of a δ-distribution with a magic angle orientation (e.g., 39°, a small angle). Therefore, the analysis we present here applies to our observation of fibrinogen adsorption. 3.4. Absolute SFG Intensities of r-Helix. Since the absolute values of the hyperpolarizability tensor elements are not available from the literature, an approximate method is used here to deduce these absolute values. The hyperpolarizability tensor for a single amide has been obtained from an ab initio molecular orbital calculation for an isolated N-methylacetamide (NMA) molecule. Specifically, we have performed a Gaussian 03 MP2 calculation with a 6-31++G** basis set. This calculation provides analytic transition dipole derivatives and numerical transition polarizability derivatives in laboratory-fixed Cartesian coordinates, which are used to deduce the desired values in normal coordinates Q. We have also considered that an average amide unit in an R-helix may be slightly different from that for a single NMA. In some early publications, the average polarizability derivatives of a peptide unit for the amide I modes of an R-helix was deduced. On the basis of their values, we have the relationships Ra′a′c′/Rc′c′c′ ≈ 0.2 and Rb′b′c′/Rc′c′c′ ≈ 0.05 for an average unit. Here (a′, b′, c′) are coordinates for the peptide unit with c′ being along the dipole transition direction of the amide I vibration.69 The angle between the axis of the R-helix and the dipole transition of an individual amide unit is ∼38°.41,69,70 Then the hyperpolarizability can be calculated by projection of each unit onto the helix-based coordinate system.41 This method has been used to calculate nonlinear hyperpolarizability for macromolecules.71 It is based on the assumption that the coupling among each unit in a long R-helix is weak. A more detailed description of this method can be found in ref 71. To address the coupling in the helix and to match the experimental IR and Raman observations, we use 42° (instead of 38°) for the angle between the axis of the R-helix and the dipole transition of an individual amide unit. We then have
βaac ) βbbc ) 4.28Rc′c′c′; βccc ) 8.14Rc′c′c′; βaca ) βbcb ) βcaa ) βcbb ) 2.62Rc′c′c′ (4) These ratios among different hyperpolarizability tensor elements agree with those deduced by the polarized IR and polarized Raman spectra.41,65-67 From the ab initio calculation we have Rc′c′c′ ∼ 1.5 × 10-26m4/Vs.
Wang et al. The absolute intensity of the amide I signal is compared to an asymmetric 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipid bilayer with a deuterated distal leaflet. The bilayer was made by the Langmuir-Blodgett/Langmuir-Schaefer method on a CaF2 substrate with a surface density of 46 Å2 per molecule. This bilayer is very stable, and the orientation has been studied.72 Since the hyperpolarizability of methyl groups has been well studied, we can use this sample to measure the absolute intensity of the protein amide I signal. The SFG intensity of the amide I signal was also compared to that of z-cut quartz.73 3.5. ATR-FTIR. ATR-FTIR has been used as a supplemental tool to examine interfacial fibrinogen orientation in this research. Data analysis for polarized ATR-FTIR spectra has been published previously40,41 and will not be repeated here. Some details of such analysis can be found in the Supporting Information along with the evaluation of the local optical field in polarized ATR-FTIR measurement in this work. 4. Results and Discussion 4.1. SFG Spectra Collection, Fitting, and Analysis. We collected SFG spectra from fibrinogen at the PS/fibrinogen solution interface in situ using different polarization combinations of the input and output laser beams. In a fibrinogen molecule, the two coiled-coils contain the majority of the R-helical components. We believe that SFG amide I signals collected from fibrinogen adsorbed at the PS/solution interface are dominated by contributions from R-helical components, and such R-helical signals are dominated by coiled-coils. To generate substantial SFG amide I signals, after fibrinogen adsorbs on the interface, it should adopt a bent conformation (Figure 1c) instead of a linear native structure.27,28 This can be confirmed by the calculated results using the computer program developed by Simpson and his colleagues.74 The calculated SFG amide I signal from R-helical structures is very weak from the native structure (no matter which orientation the molecule adopts), but the R-helical signal can be much stronger when calculating from a coiled-coil segment in certain orientations. This clearly indicates that the two coiled-coil segments in the native fibrinogen structure with an (approximate) inversion symmetry cannot generate a strong SFG signal, but a bent structure with two coiled-coils can lead to a much stronger SFG signal. In this paper, we want to quantitatively deduce the interfacial conformational and orientational ordering of fibrinogen by determining the orientation distribution of the two coiled-coils. The SFG spectra collected with different polarization combinations are shown in Figure 3a. Analyzing the vibrational spectra of protein molecules in the amide I frequency region is difficult because many peaks arising from various secondary structures can overlap in this region. Extensive FTIR studies have been carried out to obtain reliable secondary structural information from such amide I signals.37-40,75 We have fitted SFG amide I bands according to the results from FTIR studies on fibrinogen, assuming each vibrational mode has the same peak center and peak width in both SFG and FTIR spectra. Therefore, we can adopt peak center and peak width for each vibrational mode deduced from extensively studied FTIR spectra. For modes that are SFG inactive but FTIR active, the intensity should be zero in the SFG fitting. This fitting strategy should be reliable, but may still have some problems. For example, there are significant frequency overlaps between SFG signals generated from “free CdO” groups (or random coils) and R-helices. The amide I frequencies of “free CdO” groups can cover from 1645 to 1660 cm-1 depending on their chemical environments. It is almost impossible to distinguish these two
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J. Phys. Chem. B, Vol. 112, No. 7, 2008 2285 one of the vibrational modes of PS. The fitting curves for SFG spectra collected from the PS/fibrinogen solution interface are shown as solid lines in Figure 3a. We want to further confirm that fibrinogen SFG spectra are dominated by contributions from R-helical structures rather than “free CdO” groups or random coils. In order to do so, we checked the relative phase between the A mode and the E1 mode in χyyz and χzzz of the SFG amide I signals. From the detailed analysis in the Supporting Information, we know that the A mode and E1 mode have opposite phases in χyyz and χxxz, while they have the same phase in χzzz. It is feasible to check the relative phase between χyyz and χzzz measured from interfacial fibrinogen experimentally by either using polarization analy sis58,59,73,76-79 or varying input angles between the input laser beams and the surface normal to collect ppp spectra.59,76 Here we only introduce the method of collecting SFG ppp spectra by varying the input angles of the incident lasers. The SFG ppp signal intensity can be written as
|
-Lxx(ω)Lxx(ω1)Lzz(ω2) cos β cos β1 sin β2χxxz -Lxx(ω)Lzz(ω1)Lxx(ω2) cos β sin β1 cos β2χxzx Ippp ∝ +Lzz(ω)Lxx(ω1)Lxx(ω2) sin β cos β1 cos β2χzxx +Lzz(ω)Lzz(ω1)Lzz(ω2) sin β sin β1 sin β2χzzz
Figure 3. (a) SFG spectra of adsorbed fibrinogen molecules at the PS/solution interface collected with different polarization combinations. (b) SFG spectra collected with small beam angles. Dots are experimental data, and solid lines are fitting curves.
kinds of structures using linear vibrational spectroscopic techniques (e.g., FTIR) by performing peak fittings in the frequency domain. In this situation, we can use SFG polarization analysis to assign such amide I peaks. When we fitted SFG spectra collected from interfacial fibrinogen molecules, we observed that the peak center in the sps (s-polarized SFG signal, p-polarized visible input, and s-polarized infrared input) spectrum is higher than those in the ssp and ppp spectra. Such a result was confirmed by repeating the same experiment many times. According to the detailed analysis shown above, the A mode and E1 mode of R-helix vibrations behave differently in the SFG amide I signals collected using different polarization combinations. The signal contributed by the E1 mode is usually stronger than that from the A mode in the sps spectrum. The peak centers of the amide I signals contributed by the A mode (1652 cm-1) and E1 mode (1657 cm-1) vary slightly.41 Our observation of different peak centers in fibrinogen sps and ssp (or ppp) spectra clearly indicates that the SFG spectra of fibrinogen at the PS/solution interface are dominated by the contribution from R-helices. For amide I signals of random coils, such a peak shift should not be observed. Therefore, we believe (and will confirm below) that fibrinogen SFG amide I spectra are dominated by contributions from R-helical structures, and it is reasonable for us to ignore the contributions from “free CdO” groups in this spectral region. We can fit fibrinogen amide I SFG bands quite well by using peaks contributed from the A mode (1652 cm-1) and the E1 mode (1657 cm-1) of R-helices and some small peaks from β-sheet structures (1633 cm-1 and 1680 cm-1). We also find that we have to use a sharp peak centered around 1600 cm-1 to fit the spectra. The intensity of this unknown peak is relatively strong in ppp and sps spectra. We believe that this peak is not contributed from fibrinogen secondary structures but may be
|
2
(5)
where Lii(ω) is a Fresnel coefficient and local field correction factor, and β, β1, and β2 are angles of the signal, visible, and IR beams versus the surface normal, respectively. We have χxzx ) χzxx for both R-helix and “free CdO” groups. Also, in our SFG experimental geometry,
Lxx(ω)Lzz(ω1)Lxx(ω2) cos β sin β1 sin β2 ≈ Lzz(ω)Lxx(ω1)Lxx(ω2) sin β cos β1 cos β2 (6) Substituting these formulas in eq 5,
Ippp ∝ -
|
-Lxx(ω)Lxx(ω1)Lzz(ω2) cos β cos β1 sin β2χxxz +Lzz(ω)Lzz (ω1)Lzz(ω2) sin β sin β1 sin β2χzzz
|
2
(7) If the input or output beam angle is equal to the critical angle of the total internal reflection, Lxx(ω) will be zero. Therefore, for our near total reflection geometry, we have
Ippp ∝ |Lzz(ω)Lzz(ω1)Lzz(ω2) sin β sin β1 sin β2χzzz|2 (8) From this discussion we know that, if all the input angles are smaller than the critical angle of the total internal reflection, the ppp spectrum is the result of destructive interference between the χxxz and χzzz components. The detailed data analysis above indicates that the A mode has the same phase in χxxz and χzzz, while the E1 mode has the opposite phase. We can expect that the E1 mode will become stronger in the ppp spectrum if the input beam angles are smaller than the critical angle. This will result in a peak center shifting to higher wavenumbers. We should not observe this phenomenon for signals contributed from the free “CdO” groups. We collected ssp and ppp SFG spectra with much smaller input and output angles (Figure 3b). The peak center of the ppp spectrum is higher than the ssp spectrum. This result confirms our above analysis and thus we believe that SFG spectra collected from interfacial fibrinogen are dominated by the contribution of R-helical structures at the interface. This experiment has been repeated many times, and the same result
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Wang et al.
Figure 5. Analysis of the effect of experimental error on the determination of the parameters of the Gaussian distribution function. Different |χyzy,E1|/|χyzy,A| ratios varying from 0.8 to 8.0 and different distribution widths (from 0° to 90°) were plotted in the figure.
Figure 4. (a) Calculated SFG ratios between the E1 mode and the A mode of R-helices in χyzy as functions of the orientation angle θ and distribution width σ. (b) Calculated ATR-FTIR absorption ratios collected with p and s polarizations. The measured ratios from fibrinogen molecules adsorbed at the PS/solution interface are also shown in the figure as thick solid lines.
was acquired. This conclusion was further confirmed by our “polarization mapping” analysis58 (spectra are not shown). 4.2. Orientation Distribution Information of r-Helices in Interfacial Fibrinogen Deduced by SFG. We believe that all fibrinogen molecules adsorbed at the PS/solution interface would not adopt the same orientation. Therefore, instead of using a δ-distribution, we adopted a Gaussian function to describe the orientation distribution of fibrinogen R-helical coiled-coils at the interface. To deduce such a Gaussian distribution, we need at least two measurements to deduce both the average orientation angle θ0 and the angle distribution width σ simultaneously. Here we consider each R-helical coiled-coil as a unit, instead of considering all the R-helical coiled-coils in one fibrinogen molecule as a unit. From the relations regarding the measured SFG susceptibility components and orientation for R-helices, we should be able to figure out how relative intensity ratios of various peaks in the same spectra or ratios of the same peak in spectra collected using different polarization combinations are related to the orientation. It was found that such intensity ratios are redundant and can only provide one measurement for R-helix. One example for such a measurement is shown in Figure 4a to relate orientation information to the measured intensity ratio between the E1 mode and the A mode in χyzy. The experimentally determined ratio for fibrinogen coiled-coils is 1.5. We cannot deduce the orientation angle θ0 and angle distribution width σ in the Gaussian function (two parameters) simultaneously with only one measurement. From Figure 4a we know that, if we assume a δ distribution, then the angle θ0 is 39°, which is close to the “magic angle” for SFG measurements.80 As stated, it is unlikely that fibrinogen coiled-coils have a narrow or even a δ-orientation distribution. In order to get more information regarding the distribution function, as we
demonstrated in our previous publications, we additionally include an absolute intensity measurement in our experiment.59,81 With two measurements, including an intensity ratio measurement and an absolute intensity measurement, we should be able to deduce two parameters for the orientation distribution function, i.e., the orientation angle θ0 and angle distribution width σ, simultaneously. To include the absolute intensity measurement, we need to know the surface coverage of fibrinogen molecules and to deduce the absolute values of molecular hyperpolarizability tensor elements for the amide I signal of an R-helix. As mentioned above, we first determined the absolute values for a single peptide unit from ab initio molecular orbital calculation for an isolated NMA molecule.30,82 On the basis of this value, we deduced the absolute values of the R-helix amide I hyperpolarizability tensor elements. The surface coverage or density of fibrinogen adsorbed on PS has been well studied in the literature.83-85 Under our experimental conditions, such coverage is about 0.6 µg/cm2. In our previous publications, by comparing the FTIR spectra of adsorbed fibrinogen with those in the native state in solution, we showed that the R-helical secondary structure of adsorbed fibrinogen on a PS surface does not change significantly.28 This shows that the R-helical content of adsorbed fibrinogen on PS does not alter much and thus can be deduced from the fibrinogen surface coverage. We can calibrate the absolute intensity in SFG spectra collected from interfacial fibrinogen by comparing such intensity to those collected from an isotopically asymmetric lipid bilayer26 and a z-cut quartz. Previously, we demonstrated how to deduce the orientation distribution of a surface methyl group based on the intensity ratio measurement and the absolute intensity measurement.59,81 Here using the detailed analysis for an R-helix, we can deduce the orientation distribution of R-helical coiled-coils in fibrinogen on the surface. At the same time, we want to discuss the possible errors for such a deduction because some approximations have been made in our absolute intensity calculation, and some uncertainties can be induced in our various experimental procedures. Figure 5 displays both the value of |χyyz,A| and the ratio of |χyzy,E1|/|χyzy,A| as functions of the orientation angle θ0 and distribution width σ for a Gaussian distribution. The measured ratio and value are shown as a thick black line and a broken line, respectively. If we assume that this is an error-free measurement/analysis, the deduced results, the crossing point of the two lines, should be our result with measured orientation
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Figure 6. (a) Orientation distribution of R-helices in the adsorbed fibrinogen layer deduced by both SFG ratio and intensity measurements; two extreme distributions are also shown for comparison. (b,c,d) Orientation distributions deduced by the maximum entropy function based on both polarized ATR-FTIR and SFG measurements. To demonstrate the effect of experimental error on the deduced orientation distribution, additional distributions (dashed lines) were calculated using an error of (50% for 〈cos θ〉 and (20% for both 〈cos2 θ〉 and 〈cos3 θ〉/〈cos θ〉.
angle θ0 of 68° and angle distribution width σ of 45°, as shown in the figure. This orientation distribution was plotted in Figure 6a. For error analysis, we included a (20% uncertainty for the ratio measurement and (20% for the absolute intensity measurement (see more details later). Since the calculated values of hyperpolarizability for the R-helix are based on several assumptions, we also included a (50% error bar in Figure 5 to cover the possible uncertainties introduced by these assumptions. Figure 5 illustrates clearly how such experimental errors affect our deduced results regarding the orientation distributions. The hatched areas in Figure 5 show the possible combinations of θ0 and σ for the orientation distribution after including these errors. In Figure 6a we also plot the two extreme distributions after including (50% error. One extreme distribution is a Gaussian distribution with θ0 ) 65° and σ ) 35°, and another one is a very broad distribution with θ0 ) 0 and σ ) 140°. The real situation should be between the two extremes. 4.3. Orientation Distribution Information of R-Helices in Interfacial Fibrinogen Deduced by Polarized AIR-FTIR. The ATR-FTIR spectra of fibrinogen adsorbed at PS/protein solution interfaces have been collected at s and p polarizations. Unlike SFG, ATR-FTIR lacks intrinsic surface specificity, thus interfacial fibrinogen spectra should be obtained after subtracting background water bending modes, as we demonstrated in our recent publications.27,28 The protein solution concentration was 1 mg/mL, and we found that the contribution from fibrinogen in the solution to the ATR-FTIR spectra is not substantial and can be ignored. According to the detailed data analysis shown in the Supporting Information and similar to the SFG case, we can plot the calculated absorbance ratios between p polarization and s polarization of an R-helix structure as a function of orientation angle (Figure 4b). For the calculation, the orientation distribution was assumed to be a delta function. The mean square local
optical fields Ex2, Ey2 and Ez2 were calculated by the thin film approximation and assuming that the refractive indices of ZnSe, polymer, adsorbed protein film, and protein solution are 2.4, 1.45, 1.45, and 1.3, respectively.86 For a more accurate calculation, we need to use the complex values for refractive indices, and the film thickness should be considered. In the Supporting Information, we showed that a thin film approximation is good enough for the data analysis here. Our measured amide I ATR-FTIR intensity ratio of WIR,p/ WIR,s of the R-helix is 1.4. According to Figure 4b, the average orientation angle of the R-helices is around 60° from the surface normal if we assume a δ distribution. It is not very far from the “magic angle” (54.7°) of IR spectroscopic measurements, and thus no definite conclusion can be drawn. Polarized ATR-FTIR can generate only one measurement. Using this measurement alone cannot provide enough information to deduce a suitable Gaussian distribution with two parameters, which requires at least two independent measurements. Figure 4b shows that the possible angles are large, indicating that the average orientation of the R-helices in adsorbed fibrinogen molecules is more parallel to the PS surface. This conclusion was further verified by the intensity ratio between amide I and amide II signals measured in s polarized ATR-FTIR spectra. The observed intensity ratio of amide I/amide II signals of R-helices indicates that the average orientation of R-helical structures in an adsorbed fibrinogen molecule is more parallel to the PS surface than to the surface normal.41,87 4.4. Orientation Distribution of Interfacial Fibrinogen Coiled-Coils Deduced by Combining SFG and Polarized ATR-FTIR Measurements. Our above discussions show that SFG experiments can provide two independent measurements to deduce a Gaussian orientation distribution of R-helical coiledcoils in interfacial fibrinogen. The ATR-FTIR experiment can provide one independent measurement, from which we would
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expect that R-helices tend to orient toward the surface. In this section, we want to combine SFG and ATR-FTIR measurements to more accurately deduce the orientation distribution, as suggested in previous publications.81,88-92 It is necessary to further validate the orientation distribution deduced by SFG, since we made an assumption that the orientation distribution is a Gaussian distribution without any physical basis for such an assumption. Further, the calculated molecular hyperpolarizability for absolute intensity measurement may introduce some error. There are two coiled-coils in one fibrinogen molecule, and very possibly, their orientation distribution function is more complicated than a Gaussian distribution. As we discussed in our previous publication, for limited measurements, the best trial function for distribution is a maximum entropy function, which is in the form of81,93 N
f(θ) ) exp(
an cosn θ) ∑ n)0
(9)
Our ATR-FTIR results provide a measurement for 〈cos2 θ〉, and SFG can measure 〈cos θ〉 and 〈cos3 θ〉. Therefore we have
1)
∫-11 exp(a0 + a1x + a2x2 + a3x3)x1 - x2 dx
〈cos θ〉 )
∫-11 x exp(a0 + a1x + a2x2 + a3x3)x1 - x2 dx
〈cos2 θ〉 )
∫-11 x2 exp(a0 + a1x + a2x2 + a3x3)x1 - x2 dx
〈cos3 θ〉 )
∫-11 x3 exp(a0 + a1x + a2x2 + a3x3)x1 - x2 dx
(10)
where the substitution of x ) cos θ is applied for convenience. The polarized ATR-FTIR measurement shows that 〈cos2 θ〉 ≈ 0.25, while SFG measurements indicate 〈cos θ〉 ≈ 0.2 and 〈cos3 θ〉 ≈ 0.5〈cos θ〉. The orientation distribution deduced using these three values is shown in Figure 6b. It is similar to the distribution deduced by SFG measurements alone, indicating that the SFG measurements are reliable, and our assumptions are reasonable. However, this distribution is not identical to the Gaussian distribution, suggesting that including more measurements can improve the accuracy of the deduction. We also evaluated the effects of possible experimental errors on the distribution function. In Figure 6b, we varied the value of 〈cos θ〉 and fixed the values of 〈cos2 θ〉 and the ratio between 〈cos θ〉 and 〈cos3 θ〉. In Figure 6c, we varied the value of 〈cos2 θ〉 and fixed the other two values. In Figure 6d, we varied the ratio of 〈cos θ〉/〈cos3 θ〉. All the orientations are quite broad, showing that fibrinogen molecules adsorbed at the PS/solution interface do not have the same orientation, instead, they have quite varied orientation with a broad orientation distribution. As shown in our previous publication,28 adsorption to the PS surface does not induce significant secondary structure changes of fibrinogen in a short time scale. We believe that adsorbed fibrinogen molecules do not adopt a linear structure, but a bent structure instead. Otherwise, it would not be possible to obtain such strong SFG amide I signal from coiled-coils. From the distribution function deduced by combinations of SFG and ATRFTIR measurements, we can deduce the average bend angle between the two coiled-coils. The average orientation angle is ∼65° between the surface normal and coiled-coils, shown in Figure 6b. In the previous discussions we did not include any assumption regarding the bend angle between the two coiled-
coils in fibrinogen. If we assume that the two coiled-coils have the same orientation angle, then the bend angle between the two coiled-coils is twice the angle between the surface normal and each coiled-coil, and the average bend angle should then be 130 ° (Figure 1c). If we assume the adsorbed fibrinogen film is a monolayer, from the size of the fibrinogen molecule and the average bend angle obtained above, we can deduce the average of the film thickness to be ∼19 nm.94,95 The ellipsometry measurement shows that the adsorbed fibrinogen film thickness on both hydrophobic and hydrophilic surfaces is around 10-30 nm for similar experimental conditions,96 which includes our deduced value. It is interesting to see that it is possible to obtain two distributions for two coiled-coils for interfacial fibrinogen molecules for certain measured data. For example, in Figure 6d when the measured 〈cos3 θ〉/〈cos θ〉 ratio is 0.6, there are two peaks in the distribution, possibly corresponding to each of the two coiled-coils in a fibrinogen molecule (Figure 1d). For our measured data, the distribution is too broad to distinguish two distinct orientation distributions of the two coiled-coils. Time-dependent SFG studies indicate that fibrinogen exhibits structural changes after adsorbing to PS.27,28 Here we are not focused on such time-dependent studies. All the SFG and ATRFTIR spectra reported in this paper were collected immediately after contacting PS with the protein solution. It is interesting to compare the orientations of melittin in a single lipid bilayer and fibrinogen on the PS surface. It has been shown that melittin has two distinct orientations (with narrow widths) in a lipid bilayer.26 We believe that this is due to the ordered structure of the lipid bilayer, which mediates specific interactions with melittin, resulting in two orientations. PS is a polymer, and usually polymer surfaces can be quite disordered. Therefore fibrinogen molecules can adopt many different orientations and bend angles after adsorption to the PS surface due to a broad range of interactions. 5. Conclusion This research developed a method to quantify orientational and conformational ordering of protein molecules adsorbed at the solid/liquid interface in situ using fibrinogen as an example. A combination of vibrational spectroscopic techniques, including the nonlinear spectroscopic technique SFG and traditional linear ATR-FTIR spectroscopy, has been applied in the research. Our research indicates that fibrinogen molecules adsorbed on PS adopt a bent conformation. The R-helical coiled-coils exhibit a broad orientation distribution. This broad distribution is caused by adsorbed fibrinogen molecules adopting varied conformations, e.g., different angles between two coiled-coils, or different orientation of fibrinogen with a similar conformation. Perhaps both effects play roles in determining such a broad distribution, but we cannot distinguish these two effects in the experiments. Quantitative information on the ordering of protein adsorbed on surfaces helps one understand protein-surface interactions in more detail. Acknowledgment. This work is supported by the Beckman Foundation and the Office of Naval Research (Grant N0001402-1-0832). We thank Professor Garth Simpson and Dr. Andy Moad at Purdue University for providing their calculated results on fibrinogen. Supporting Information Available: More details regarding the SFG theory and data analysis, analysis of the hyperpolarizability of R-helices, polarized ATR-FTIR data analysis, and
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