J. Phys. Chem. B 2006, 110, 5017-5024
5017
Vibrational Spectroscopic Studies on Fibrinogen Adsorption at Polystyrene/Protein Solution Interfaces: Hydrophobic Side Chain and Secondary Structure Changes Jie Wang, Xiaoyun Chen, Matthew L. Clarke, and Zhan Chen* Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed: June 26, 2005; In Final Form: October 6, 2005
Structural changes of fibrinogen after adsorption to polystyrene (PS) were examined at the PS/protein solution interface in situ using sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Different behaviors of hydrophobic side chains and secondary structures of adsorbed fibrinogen molecules 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.
1. Introduction In the last few decades, substantial progress has been achieved in the determination of native protein structures because of advances in molecular simulations and various experimental techniques such as X-ray crystallography, NMR, and mass spectrometry.1-5 However, the above-mentioned experimental techniques have difficulties determining molecular structures of interfacial proteins, and simulated results require experimental data to ensure their accuracy. Behaviors of proteins on surfaces and at interfaces play important roles in many biological activities.6-11 For example, the adsorbed protein layer on a biomaterial surface determines the material’s biocompatibility,6 and interactions between adhesive proteins and antibiofouling coatings for marine vessels control whether such coatings are effective.7 To date, the deduction of structural information such as orientation and conformation of proteins at interfaces is still quite challenging. Linear vibrational spectroscopy techniques such as attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) have been used to study protein conformation at interfaces for decades.12-14 By fitting these vibrational spectra in detail, especially in the protein amide band range, important information regarding structures of interfacial proteins such as the relative percentage of each protein secondary structure can be measured. Polarized ATR-FTIR can also provide some information about the orientation of these secondary structures. However, the information provided by ATR-FTIR is often not adequate for the understanding of interfacial protein structures. Recently, a second-order nonlinear vibrational spectroscopic technique, sum frequency generation (SFG) vibrational spectroscopy, has been applied to elucidate protein structures at various interfaces in situ.15-33 Nonlinear spectroscopy has the potential to provide more structural information of samples under study than linear spectroscopy techniques. For example, the second-order nonlinear susceptibility tensor, which is the * To whom correspondence should be addressed. E-mail: zhanc@ umich.edu. Fax: 734-647-4685.
detected property of a second-order nonlinear optical process, can have as many as 27 independent tensor elements, depending on the symmetry of the sample. Thus, more measurements can be achieved using nonlinear optical techniques such as SFG. In addition, nonlinear spectroscopy can measure different parameters compared to linear spectroscopy,34-36 and from results obtained using both linear and nonlinear spectroscopy, more detailed structural information of proteins at surfaces/ interfaces can be deduced. As demonstrated in the literature, SFG is a versatile nonlinear optical vibrational spectroscopic technique with excellent surface sensitivity, which not only permits the identification of surface species but also provides chemical structural information such as the orientation and orientation distribution of chemical groups at interfaces or within thin films.37-48 We adopted the thin film model to analyze SFG spectra collected from interfacial proteins.44 We believe that SFG spectra collected from the polymer/protein solution interface can be correlated to the structure of the entire adsorbed protein molecules, rather than being limited to part of the protein. The detailed analysis of these protein SFG spectra collected in the C-H and O-H stretching regions demonstrates that proteins adsorbed on different surfaces have varied structural or orientational changes.15,16 We also found that the changes in SFG C-H signal collected from adsorbed proteins depend more on the contacting surface hydrophobicity and protein surface coverage than on the bulk protein solution pH.15,16 Recently, SFG amide I signals from interfacial proteins have been detected at the solid/liquid interface in situ,22,23 showing the feasibility of obtaining detailed protein secondary structural information at interfaces from SFG measurements. Most methyl, phenyl, and methylene groups are located on the hydrophobic side chains of protein molecules. Therefore, SFG signals in the C-H stretching region are dominated by side chain contributions and can be used to probe structures of protein hydrophobic side chains at interfaces.15,16 In contrast, SFG amide I signals are indicators of protein secondary structures.22-25 Therefore, we believe that SFG studies on interfacial proteins in both C-H stretching and amide I
10.1021/jp0534683 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/09/2006
5018 J. Phys. Chem. B, Vol. 110, No. 10, 2006 spectral regions will be able to provide more structural information on adsorbed protein molecules. In this paper, we investigated hydrophobic side chain and secondary structure changes of proteins after adsorption on a polymer surface by examining SFG spectra in both the C-H stretching and amide I regions. We used fibrinogen as a model protein and polystyrene (PS) as a model polymer in this research. In addition to the SFG technique, ATR-FTIR has also been used as a supplemental tool to elucidate structural changes of fibrinogen after adsorption. 2. Experimental 2.1. Sample Preparation. Bovine fibrinogen was purchased from Sigma and was used as received. Fibrinogen solution was made by dissolving fibrinogen in phosphate buffered saline (PBS) with a total ionic strength of ∼0.14 M and a pH value of 7.4. The PBS was made using deionized water (18.2 MΩ cm) from a Millipore ultrapure water system. PS and deuterated PS (d-PS) samples were purchased from Scientific Polymer Products Inc. For SFG measurements, the PS and d-PS films were made by spin coating a 2 wt % polymer solution in toluene onto IR grade fused silica or CaF2 prism substrates (ESCO products) at 2500 rpm. The fused silica prisms were cleaned thoroughly by storing them in a potassium dichromate/sulfuric acid solution for 24 h and heating them in the same solution for about 30 min. The prisms were then rinsed repeatedly using deionized water and finally dried in a dry N2 stream. The CaF2 prisms were cleaned by air plasma and rinsed thoroughly by toluene before spin coating to ensure that the surface was free of contamination. For ATR-FTIR measurements, the ZnSe ATR crystal was cleaned by toluene and polymer films were solventcast onto the crystal. 2.2. ATR-FTIR Experiments. The theory and experimental details of ATR-FTIR have been extensively published and will not be repeated here.12-14 The ATR-FTIR experiments reported in this paper were conducted using a Nicolet 550 FTIR spectrometer with a standard 45° ZnSe ATR cell. The thickness of a PS film on the ZnSe crystal can be determined by measuring the ATR-FTIR spectral intensity of the film. We used PS films with similar thicknesses for all experiments. In an ATR-FTIR experiment, PBS solution was brought into contact with the PS film for at least 2 h to ensure that the polymer film was in an equilibrated state. Then, a background spectrum of the PS contacting PBS was collected. The PBS solution was replaced by fibrinogen solution, and a new ATRFTIR spectrum of PS contacting protein solution was recorded. The interfacial protein spectrum was obtained by subtracting the background spectrum from this new spectrum. To evaluate the possible signal contributions from proteins in the solution, the protein solution was replaced by PBS solution and another ATR-FTIR spectrum was collected from the PS (with adsorbed fibrinogen)/PBS buffer interface. No substantial spectral changes were observed. Therefore, we believe, that for the fibrinogen solutions used in this research, fibrinogen molecules in the solution bulk do not substantially contribute to the ATR-FTIR spectra collected from the PS/fibrinogen solution interface and such spectra are dominated by contributions from adsorbed fibrinogen molecules. 2.3. SFG Experiments. SFG theory has been extensively published and will not be repeated here.37-48 Previous research indicated that SFG can selectively probe protein molecules on a surface or at an interface, without contributions from the protein molecules in the bulk solution.15,16 The SFG setup in our lab has been described in detail in our previous publica-
Wang et al. SCHEME 1. Trinodular Structure of Fibrinogen with Labeled Domains
tions.42 All the SFG spectra shown in this paper were collected from adsorbed fibrinogen molecules at the PS/fibrinogen solution interface with the near total internal reflection geometry.22 SFG spectra with ssp (s-polarized output, s-polarized visible input, and p-polarized IR input) and ppp polarization combinations were collected. 2.4. Quartz Crystal Microbalance Measurements. In this research, a quartz crystal microbalance (QCM) has also been used as a supplemental technique to monitor the time-dependent adsorption amount of fibrinogen on PS and to compare the adsorption amounts when different concentrations of fibrinogen solution were used. The QCM equipment used in this study was purchased from MAXTEK Inc. First, 10 MHz quartz crystals (Universal Sensors) were rinsed repeatedly using toluene. Then PS films were made by spin coating a 2 wt % PS solution in toluene on these quartz crystals. The relative fibrinogen adsorption amounts were compared by assuming a linear relationship between the crystal frequency change and the adsorbed mass change.49 3. Results and Discussion 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-coil domains.17,50-53 The three hydrophobic domains are two outer D domains and one central E domain. Each D domain is connected by a coiled-coil segment to the central E domain (Scheme 1). Therefore, a native fibrinogen molecule possesses more or less an inversion center, showing that fibrinogen in the native state may not contribute strong SFG signals in the amide I region from the two coiled-coils. Research shows that adsorbed fibrinogen on a surface can change its conformation from the native structure and the conformation of surface-bound fibrinogen plays an important role in thrombus formation.17 However, details of such conformations on various surfaces are not known. In this research, we hope to elucidate structural changes of fibrinogen after adsorption on the PS surface. 3.1. Hydrophobic Side Chains of Adsorbed Fibrinogen. Figures 1 and 2 show SFG spectra collected from the d-PS/ fibrinogen solution interface in the C-H and O-H stretching spectral region with protein solution concentrations of 0.005 and 1 mg/mL, respectively. SFG spectra of d-PS/air and d-PS/ PBS solution interfaces were collected before contacting polymer surfaces with protein solutions (spectra not shown). No C-H signals were detected in such spectra, indicating that the polymer surfaces were free from hydrocarbon contamination. Then, the d-PS surface was brought into contact with a protein solution, and SFG spectra were immediately collected from the d-PS/fibrinogen solution interface. SFG spectra collected in the C-H stretching region from the 1 mg/mL fibrinogen sample exhibit almost no time-dependent change; they became relatively stable within 1 min after contact. It took a much longer time for the 0.005 mg/mL fibrinogen solution sample to generate stable SFG spectra from the d-PS/solution interface. This result is compatible with those published in the literature, which showed that protein adsorption on a surface from a lower protein
Fibrinogen Adsorption at Solution Interfaces
Figure 1. SFG spectra collected from the interface between PS and 0.005 mg/mL fibrinogen solution in the C-H stretching region: upper panel, ppp spectrum; lower panel, ssp spectrum (the upper spectrum is offset for clarity). The dots are experimental data. The solid line in each spectrum overlapping the dots is the overall fitting. The other solid lines are fitting components contributed by different chemical groups. The magnitude of the fitted phenyl peak at ∼3050 cm-1 is multiplied by 10.
Figure 2. SFG spectra collected from the interface between PS and 1 mg/mL fibrinogen solution in the C-H stretching region: upper panel, ppp spectrum; lower panel: ssp spectrum (the upper spectrum is offset for clarity). The dots are experimental data. The solid line in each spectrum overlapping the dots is the overall fitting. The other solid lines are fitting components contributed by different chemical groups. The magnitude of the fitted phenyl peak at ∼3050 cm-1 is multiplied by 10.
solution concentration requires a much longer time to reach equilibrium or to maximize the protein surface coverage.54-56 The SFG spectra shown in Figure 2 were collected when the SFG spectra became relatively stable, about 30 min after the protein solution contacted the d-PS. For comparison, the SFG spectra shown in Figure 1 were also collected about 30 min
J. Phys. Chem. B, Vol. 110, No. 10, 2006 5019 after contact. As mentioned, such spectra are very similar to those collected immediately after contact. At longer contacting times between d-PS and both protein solutions, further minor and slow spectral changes were observed. These long-term changes will be ignored in the current analysis and will be discussed later in the paper. The SFG spectra in Figures 1 and 2 were fitted by the method published in the literature,15 and the fitting results are also shown in the figures. Peak assignments for hydrophobic side chains of proteins in SFG spectra have been extensively discussed.15,16 The peaks at 2870, 2935, and 2965 cm-1 can be assigned to the symmetrical stretching vibration, Fermi resonance, and asymmetric stretching vibration of methyl groups, respectively. The peaks at 2850 and 2920 cm-1 are assigned to stretching modes of methylene groups. The peaks near 3050 cm-1 are contributed by vibrational modes of phenyl groups. These methyl, phenyl, and methylene groups are mainly located in the hydrophobic side chains of protein molecules. Therefore, SFG signals in the C-H stretching region can be used to probe structures of hydrophobic side chains of protein molecules at interfaces. Our previous SFG studies on various interfacial proteins demonstrated that hydrophobic side chains of adsorbed protein molecules at the contacting media/protein solution interface tend to align toward the hydrophobic contacting surface.15,16 We believe this is also the case for the fibrinogen molecules studied here, and we will confirm this later in the paper. The d-PS surface is quite hydrophobic with a water contact angle of about 90°. Therefore, it should induce a quite ordered hydrophobic side chain alignment from fibrinogen molecules adsorbed at the d-PS/fibrinogen solution interface. This was confirmed by the fact that strong SFG signals can be detected from hydrophobic groups of fibrinogen at the interfaces (Figures 1 and 2). It is interesting to observe that the SFG intensities of C-H stretching signals collected from the interface between d-PS and fibrinogen solutions with two different solution concentrations are similar. SFG signal intensity is related to the orientation of chemical groups and is proportional to the square of the group density (or coverage of the group at the interface). To understand the detailed structural differences between hydrophobic side chains of adsorbed fibrinogen at the two interfaces with different solution concentrations, we should quantify the orientation distributions of C-H groups (e.g., methyl groups) of specific hydrophobic side chains on the interfacial fibrinogen at these two interfaces, which can be addressed using site-specific isotope-labeled proteins.57 Such a study is beyond the scope of this article. Here, we will perform some qualitative deduction rather than quantitative analysis. We measured the relative adsorption amounts of fibrinogen from the two solution concentrations on PS using QCM. The QCM results indicate that the maximal adsorption amount of adsorbed fibrinogen from the 1 mg/mL solution on d-PS is about three times higher than that from the 0.005 mg/mL solution. If the adsorbed fibrinogen molecules at the two interfaces adopt the same structure, the spectral intensity of fibrinogen at the d-PS/1 mg/mL fibrinogen solution should be about nine times stronger. Figures 1 and 2 clearly indicate that this is not the case; the SFG spectral intensities in Figures 1 and 2 are not very different. According to the thin film model, this shows that on average each adsorbed fibrinogen molecule from the 0.005 mg/mL solution must contribute more to the SFG signal. That is to say, it has to expose more C-H groups to the d-PS surface. Therefore, similar SFG C-H signal intensities collected from interfaces between d-PS and fibrinogen solutions with different concentrations demonstrate that different structures of
5020 J. Phys. Chem. B, Vol. 110, No. 10, 2006
Figure 3. SFG spectra collected from the interface between CaF2 and 1 mg/mL fibrinogen solution in the C-H stretching region: upper spectrum, ppp spectrum; lower spectrum, ssp spectrum (the upper spectrum is offset for clarity).
fibrinogen exist at the two interfaces. For the dilute solution, more side chains in each adsorbed protein can orient toward the surface, because the each adsorbed protein has more room and the surface-protein interaction is dominant. While for the high concentration solution, more proteins adsorb at the interface, and protein-protein interactions play a stronger role, resulting in less hydrophobic side chains orientated toward the surface for each fibrinogen molecule. In addition to the spectral intensity, there are also some differences in the spectral features of the SFG spectra collected from these two interfaces, further confirming that the orientation and orientation distribution of the hydrophobic side chains in each adsorbed fibrinogen are different at the two interfaces. This result is consistent with our observations of structural differences of adsorbed bovine serum albumin (BSA) molecules at the air/solution interfaces with BSA solutions of different concentrations.54 To further confirm our conclusion that the ordered orientation of hydrophobic side groups of adsorbed fibrinogen at the d-PS surface/protein solution interface is induced by the favorable hydrophobic interactions between these groups and the hydrophobic d-PS surface, we replaced the d-PS surface with a CaF2 surface and repeated the SFG experiments. The CaF2 surface is much more hydrophilic compared to the d-PS surface. Figure 3 shows the SFG spectra collected from fibrinogen adsorbed at the CaF2/fibrinogen solution interface in the C-H and O-H stretching region. Unlike those collected from the d-PS/solution interface, here no C-H stretching signal was detected. Because the CaF2 surface is hydrophilic, it does not induce preferential alignment of hydrophobic groups toward its surface, thus no SFG signal was detected. Similar phenomena have been observed from other protein molecules adsorbed on other hydrophilic surfaces.15-17 Besides the C-H stretching signals, SFG spectra collected from solid/protein solution interfaces shown in Figures 1-3 also contain significant contributions from O-H and N-H stretching modes. According to our analysis in ref 15, the O-H stretching signals are dominated by the interfacial water molecules, which were ordered by charged interfacial proteins. The N-H stretching signals can be contributed by the side chains of some amino
Wang et al.
Figure 4. ATR-FTIR spectra (a) from the interface between the PS surface and 1 mg/mL fibrinogen solution, (b) from the interface between the PS surface and 0.005 mg/mL fibrinogen solution, and (c) from fibrinogen in the bulk solution. The band between 1500 and 1600 cm-1 is the amide II signal, and the band between 1600 and 1700 cm-1 is the amide I signal (the a and b spectra are offset for clarity). Experimental data and overall fitting results are shown as lines.
acid residues in the protein and/or the protein backbone, which will be discussed in more detail later in the paper. 3.2. Secondary Structural Changes of Adsorbed Fibrinogen Molecules. The SFG measurements on the C-H groups discussed in the previous section indicate that each adsorbed fibrinogen molecule at the d-PS/fibrinogen solution interface contributes more SFG signal if the solution had a lower concentration. This shows that the hydrophobic C-H groups in adsorbed fibrinogen at this interface undergo more substantial orientational changes than those at the interface with a higher solution concentration. In this section, we will study fibrinogen secondary structures at the d-PS/fibrinogen solution interface using SFG supplemented by ATR-FTIR. Compared to hydrophobic side chains, secondary structural changes of protein molecules usually represent more global protein structural changes. Here, we will first present the ATR-FTIR results, followed by SFG studies on amide I signals. 3.2.1. ATR-FTIR Studies. It has been extensively demonstrated that protein vibrational spectra in the amide I region can provide abundant structural information about protein secondary structures.58-60 ATR-FTIR has been widely applied to investigate amide I spectra of adsorbed proteins. In the ATR-FTIR measurements here, as with the previous SFG C-H studies, two fibrinogen solutions with concentrations of 1 and 0.005 mg/ mL are used. The ATR-FTIR spectra of adsorbed fibrinogen at the PS/protein solution interfaces are shown in Figure 4. As mentioned in section 2.2, these ATR-FTIR spectra are mainly contributed by the adsorbed fibrinogen molecules at the interfaces. ATR-FTIR results shown in Figure 4 indicate that the coverage of adsorbed fibrinogen on the d-PS surface using the 1 mg/mL fibrinogen solution is about three times higher than that from the lower concentration. This result agrees with that obtained from the QCM measurement. More detailed
Fibrinogen Adsorption at Solution Interfaces understanding of fibrinogen adsorbed at these two interfaces can be derived by fitting the ATR-FTIR spectra. Spectral fitting for protein FTIR spectra in the amide region has been extensively discussed in the literature.12,58-61 Various functions such as Gaussian, Lorentzian, and Voight functions have been used in such fittings. We found that similar results can be obtained when we fit the ATR-FTIR spectra collected using different functions, thus here we will show the results fitted using a Lorentzian function. Vibrational spectra of fibrinogen molecules have been widely studied, and various vibrational peaks have been assigned.62-64 Here, we fit the ATRFTIR amide I spectra using the following peak assignments: the 1630 and 1685 cm-1 peaks for β-sheet structures, the 1653 cm-1 peak for R-helices, and the 1645, 1665, 1675 cm-1 and remaining peaks for random coils and turns. The ATR-FTIR spectra collected from adsorbed fibrinogen molecules with different coverages at the PS/fibrinogen solution interface do not have significant spectral feature differences. More interestingly, these ATR-FTIR spectra of adsorbed fibrinogen are quite similar to the spectra collected from fibrinogen molecules in solution. Details about how to collect vibrational spectra of fibrinogen in bulk solution using ATRFTIR can be found in the literature.65 Briefly, an ATR-FTIR spectrum of fibrinogen solution with high protein (∼20 mg/ mL) concentration was collected first. Then the protein solution was replaced by PBS. The FTIR spectrum of fibrinogen in solution was obtained by subtracting the second spectrum from the first spectrum. The ATR-FTIR spectra shown in Figure 4 collected with different bulk concentrations have amide I spectra very similar to fibrinogen in bulk solution, which is a markedly different result from the SFG C-H results observed from the hydrophobic side chains of adsorbed fibrinogen. Those SFG C-H results indicate that substantial orientational changes of protein hydrophobic side chains occurred immediately after adsorption. This indicates that the orientation changes of hydrophobic side chains do not significantly influence the secondary structures on this short time scale (immediately after adsorption). We hypothesize that the different results observed from SFG C-H stretching signals of hydrophobic side chains and ATRFTIR spectra from protein secondary structures after adsorption are due to the differences in the scale and range of these changes: the orientation change of hydrophobic side chains can be quite local, and secondary structural changes can be quite global or at least not very local. The local side chain orientation changes do not significantly affect the secondary structure. This hypothesis will be further proved by the later experiments. We should point out here that although no significant spectral feature changes were found in the ATR-FTIR spectra collected from adsorbed fibrinogen in the amide I range, there are still some minor spectral feature changes. The similarity between ATR-FTIR amide spectra of bulk solution fibrinogen and adsorbed fibrinogen at the two interfaces and the lack of substantial time-dependent ATR-FTIR amide I spectral changes of fibrinogen after adsorption indicate that no substantial secondary structural changes occurred for fibrinogen after adsorption to PS. For example, no considerable changes in the amount of different secondary structural components, such as R-helices, β-sheets, and various turns, happened after fibrinogen adsorption. 3.2.2. SFG Studies. Different from ATR-FTIR spectra, SFG spectra selectively probe the interfacial fibrinogen; it is not necessary to subtract background water bending signals. Our previous research also demonstrated that proteins in the solution
J. Phys. Chem. B, Vol. 110, No. 10, 2006 5021
Figure 5. SFG spectra in the amide I region collected from the interface between the PS surface and fibrinogen solution with a concentration of 1 mg/mL (left) and 0.005 mg/mL (right). The dots are experimental data. The solid line in each spectrum overlapping the dots is the overall fitting. The other solid lines are individual peaks. Their assignments are described in the text.
bulk will not contribute SFG signals.15,16,22 SFG amide I spectra were collected from adsorbed fibrinogen at the interface between PS and fibrinogen solutions with the above two solution concentrations. Such spectra change as a function of time. Figure 5 shows the ssp spectra with the maximal intensities in each case. These two SFG spectra were fitted using Lorentzian functions with parameters (ωq and Γq) derived from our above ATR-FTIR spectral fitting results. SFG fitting peaks are plotted as solid lines in Figure 5. Figure 5 clearly shows that the maximal intensity of the SFG spectrum in the amide I range collected from fibrinogen adsorbed at the interface between PS and the 1 mg/mL fibrinogen solution is about 10 times stronger than that with the lower concentration. This is very different from the conclusion derived from SFG spectra collected in the C-H stretching region (Figures 1 and 2). It is also interesting to see that no amide II signal has been detected by SFG. This agrees very well with theory, since under the dipole approximation, only modes that are both IR and Raman active can be detected by SFG. Similar to the C-H stretching region, we also collected SFG spectra from adsorbed fibrinogen at the CaF2/fibrinogen solution interface in the amide I region. As discussed in section 3.1, no SFG signal from C-H groups was detected at this interface (Figure 3). Differently, in the amide I region, SFG signal can be detected with a strong intensity (Figure 6). This also clearly indicates that the adsorption order, orientation, and conformation of protein secondary structures at solid/solution interfaces can be very different from hydrophobic side chains. Now, we want to elucidate more detailed time-dependent structural changes of fibrinogen after adsorption to the PS surface. From the interface between PS and the 1 mg/mL fibrinogen solution, both SFG C-H stretching and amide I signals were immediately observed. The amide I signal reaches its maximum intensity within 1 min, then it decreased gradually as a function of time. The ssp SFG amide I intensity of adsorbed fibrinogen decreased about 30% after 2 h. For this fibrinogen solution concentration, the QCM results show that adsorbed fibrinogen maintains a similar mass after adsorption as a function of time, indicating that time-dependent changes of the protein amide I signal after adsorption to PS are not due to changes in the protein adsorption amount. We believe that this time-dependent SFG amide I signal intensity change is related to the slow orientation and conformation changes of adsorbed fibrinogen, especially
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Figure 6. SFG spectra collected from the interface between the CaF2 surface and 1 mg/mL fibrinogen solution in the amide I region: upper panel, ppp spectrum; lower panel, ssp spectrum (the upper spectrum is offset for clarity).
for changes related to secondary structures. On the contrary, there is almost no time-dependent C-H signal change after 1 min of adsorption. Such hydrophobic side chain changes can reach a near “equilibrium” shortly after adsorption, because these changes are local changes and only minor future time-dependent structural changes of such side chains were detected. Differently, changes related to secondary structures of fibrinogen observed in the amide I region are much slower. It took a much longer time (about 30 min) for both the C-H stretching and amide I signals to reach their highest intensities in the SFG spectra collected from fibrinogen at the PS/0.005 mg/mL fibrinogen solution interface. About 30 min after adsorption, the C-H stretching signals remained more or less the same but the SFG amide I signal started to decrease gradually as a function of time. The QCM measurement results indicate that, at the lower fibrinogen concentration, it took about 30 min for fibrinogen to reach its adsorption equilibrium on the PS surface. Therefore, after the PS contacted fibrinogen solution, both C-H and amide I signals increased, indicating more adsorption of fibrinogen at the interface. After the maximal adsorption, the C-H signals remain almost the same, showing that hydrophobic side chains have no significant further structural changes after adsorption. On the contrary, fibrinogen amide signals start to decrease, due to the post-adsorption structural changes related to secondary structures of interfacial fibrinogen. According to Scheme 1, if a fibrinogen molecule adopts a linear structure, then it has an approximate inversion symmetry. Therefore, such a molecule would not generate strong amide I signal. We believe that, after adsorption on the PS surface, fibrinogen would not adopt such a linear structure, because a strong amide I signal can be detected using SFG. This strong amide I signal is centered at 1650 cm-1, indicating that the signal is mainly contributed by R-helical components.17,50-53 Since, in a fibrinogen molecule, the coiled-coils contain the majority of the R-helical components, we believe that the amide I signals are dominated by signals generated from such coiled-coils. This
Wang et al. has been confirmed by evaluating the absolute intensity of the SFG signal of R-helices in fibrinogen and the relative intensity which can be generated from the coiled-coils vs R-helices in other parts of the fibrinogen molecule using a computer program designed by Garth Simpson and co-workers.66 To generate strong SFG amide I signals, fibrinogen molecules would need to adsorb on the surface with a bent structure. Then due to surface-protein interactions, such a bent structure would either gradually change into a linear structure or gradually lay down on the surface, evidenced by the decrease of the SFG amide signal as a function of time. For fibrinogen adsorbed onto the PS surface from a high protein concentration, the adsorbed protein amount reaches equilibrium very fast. Next, a post-adsorption change occurs, evidenced by the slow intensity decrease of the amide I signal. The time-dependent behavior of the amide I signal change from fibrinogen adsorbed from a lower concentration solution is the overall effect from two processes: (1) more fibrinogen adsorbing on the surface and (2) bent structures of adsorbed fibrinogen changing into linear structures or lying down. The first process enhances the spectral intensity, and the second process decreases the spectral intensity. The overall trend of the amide I intensity shows an initial increase and subsequent decrease. Thus, the time for amide I signals to reach their maximum is slightly different from C-H signals. In addition to the detection of C-H stretching signals and amide I signals, O-H signals from water molecules (3200 and 3400 cm-1) and N-H stretching signals (3270 cm-1) from adsorbed fibrinogen can also be observed from fibrinogen at the PS/solution interface. The water structure at the interface is beyond the scope of this article and will not be discussed here. The time-dependent spectral intensity changes of the N-H stretching signal correlate quite well to those of the amide I SFG signals. The stronger the amide I signal, the stronger the N-H stretching signal. Such a correlation suggests that the N-H signal in SFG spectra may be dominated by the amine groups on the peptide backbone, but we cannot exclude the possibility that side chain N-H groups may also contribute SFG N-H signals. Different behaviors of SFG C-H stretching signals and amide I signals show that fibrinogen hydrophobic side chains and secondary structures have different responses to the PS surface. The PS surface is quite hydrophobic, which can induce significant orientation changes of adsorbed protein side chains. Such hydrophobic side chain changes can be quite local and do not necessarily induce immediate changes to the protein secondary structures. Indeed, protein hydrophobic side chains are usually very flexible and can undergo local orientation changes without significantly affecting the protein secondary structure. We believe that such hydrophobic side chain structural changes of adsorbed proteins are mainly mediated by the “hydrophobic interaction.” When two “hydrophobic” or nonpolar media in water approach each other, they interact with each other through van der Waals interactions. In addition, their surface areas exposed to water are reduced, resulting in a change of hydrogen bonding environment and the entropy of water. Both van der Waals and hydrogen bonding interactions are quite local, and the dimensions of protein secondary structures are usually much bigger than the ranges of these two forces. Therefore, we believe that, at least initially, the change (e.g., orientation change) of protein secondary structures should depend more on the long-range interactions such as forces induced by interfacial electric fields. More detailed results on the interfacial electric field induced orientation change of protein
Fibrinogen Adsorption at Solution Interfaces secondary structures will be reported in a separate article.67 The structural rearrangement of adsorbed protein molecules are mediated by competition between long-range and short-range forces. The immediate structural changes of hydrophobic side chains upon adsorption are controlled by the short-range forces, while long-range forces affect the initial orientation of the entire protein and secondary structures. Later, short-range forces, such as hydrophobicity interactions, can gradually change the orientation and conformation of protein secondary structures. As the protein global conformation changes, additional hydrophobic side chain changes can be induced, but these are not as obvious as the initial adsorption changes. As mentioned, our SFG C-H stretching signals have some minor and slow changes as a function of time after reaching “equilibrium,” indicating such long-term hydrophobic side chain changes do occur. Our research results suggest that surface hydrophobicity (reported here) and surface charge (to be reported) have different effects on the orientation and conformation of adsorbed protein molecules. When both surface hydrophobicity and surface charge are controlled, it is possible to control the orientation and conformation of adsorbed protein molecules. Further investigations are being carried out by step wise modification of polymer surface hydrophobicity and charge density. We have investigated secondary structural changes of fibrinogen adsorbed on other polymer surfaces, including a polyurethane, a fluorinated polymer, and a silicone polymer using SFG. At different surfaces, fibrinogen adopted different post-adsorption secondary structural changes.68 To elucidate molecular structures of interfacial proteins, we believe that it is important to examine SFG spectra of interfacial proteins in the amide I spectral region. Such research is still in its infancy, and further detailed quantitative analysis will be reported in the future. Summary Fibrinogen adsorption on PS was examined using SFG supplemented by ATR-FTIR. Our SFG studies in the C-H stretching range indicate that fibrinogen side chains have immediate structural changes after adsorption. ATR-FTIR studies show that no substantial amount of secondary structural conversions occurred after fibrinogen adsorption. SFG amide I spectra indicate that, after the initial adsorption, fibrinogen adopted a bent structure. Such a bent structure changed slowly into a linear structure or lay down structure. This conformational change is much slower compared to the side chain structural changes of fibrinogen after adsorption. Such knowledge of structural changes of fibrinogen on surfaces should lead to an in-depth understanding of platelet adhesion and the blood compatibility of surfaces. This research demonstrated that SFG is a powerful technique to provide molecular level information of protein structural changes at the solid/liquid interface in situ and verified the importance of the detection of SFG amide I signals. SFG C-H signals can be used to elucidate local side chain structural changes, and SFG amide I signals can be used to deduce global conformation changes of proteins after adsorption. Acknowledgment. This work is supported by the Beckman Foundation, Office of Naval Research (N00014-02-1-0832) and National Science Foundation (CHE-0315857). M.C. thanks the Rackham Graduate School of the University of Michigan for a Rackham Predoctoral Fellowship. References and Notes (1) Wu¨thrich, K. Angew. Chem., Int. Ed. 2003, 42, 3340-3363. (2) Sigler, P. B.; Xu, Z. H.; Rye, H. S.; Burston, S. G.; Fenton, W. A.; Horwich, A. L. Annu. ReV. Biochem. 1998, 67, 581-608.
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