Heterodyne-Detected Achiral and Chiral Vibrational Sum Frequency

Apr 22, 2015 - Achiral spectra also showed a significant change. ... the improvement of the signal-to-noise ratio by using the heterodyne detection wa...
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Heterodyne-Detected Achiral and Chiral Vibrational Sum Frequency Generation of Proteins at Air/Water Interface Masanari Okuno and Taka-aki Ishibashi* Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan S Supporting Information *

ABSTRACT: We present complex achiral and chiral vibrational sum frequency generation (VSFG) spectra at the air/ water interface of protein solutions by using heterodynedetected VSFG. Bovine serum albumin, pepsin, concanavalin A, and α-chymotrypsin were measured as model proteins. The obtained achiral Im[χ(2)] spectra gave us insights into the molecular orientation of protein molecules and water at the interface. From the chiral Im[χ(2)] spectra in the NH stretching and amide I regions, the secondary structures of the interfacial proteins were deduced. We attributed the chiral signals in the amide I and NH stretching regions to the interface on the basis of the phase of the signals. All the achiral and chiral spectra in each region showed the same sign despite different secondarystructure contents of the examined proteins. Real-time observation of the spectral change of α-chymotrypsin was also performed by heterodyne-detected chiral VSFG. The signal intensity of the chiral Im[χ(2)] spectra in the NH stretching and amide I regions decreased on the scale of 10 min, originating from the decrease of the portion of antiparallel β-sheet conformation in the molecule. The conformational change occurred not in the bulk but at the interface. Heterodyne-detected achiral and chiral VSFG are capable of addressing the molecular orientation and conformation of proteins at air/water interfaces.

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the second-order nonlinear susceptibility, χ(2).13−15 The phase carries the information on the polar molecular orientation. Contrastingly, in the conventional homodyne detection, the valuable phase information is lost because the square of the susceptibility, |χ(2)|2, is detected. Air/water interface and lipid/water interface have been investigated using heterodyne-detected (HD) VSFG, and detailed information on the molecular configuration in the systems was collected.16−21 Now, even two-dimensional VSFG has been developed to obtain information on the dynamics of interfacial molecules.22−24 In particular, it was shown by 2D-IR in bulk and 2D-HD-VSFG that a peptide at the surface showed different dynamics, strongly indicating the environmental difference between bulk and the surface.25 Thus, HD-VSFG spectroscopy is used as a powerful probe of molecules at interfaces. Furthermore, VSFG spectroscopy is now attracting much attention as a means to detect molecular chirality.26 Thanks to its high sensitivity, chiral VSFG has emerged as a tool for investigating molecular chirality at interfaces or thin films. As a chiral probe, vibrational circular dichroism (VCD) is often employed for studying proteins and peptides in the solution

INTRODUCTION In living organisms, most of the intracellular enzymic processes occur at phase boundaries, that is, the membrane−water interface. At interfaces, protein molecules change their conformation and molecular orientation from those in bulk and then function properly. Understanding the molecular functionality of proteins in living systems requires gaining information not only on bulk but also on interfacial properties. In technological applications, the characterization of interfacial proteins at the molecular level is essential for developing functioning biomaterials such as biocatalysis and biosensors. Thus, determination of secondary structure and molecular orientation of proteins at interfaces is highly important to understand and control the function of proteins. However, there are few methods that investigate interfacial proteins in situ without staining,1−4 while the tools for studying their properties in bulk have been well established. Vibrational sum frequency generation (VSFG) spectroscopy is suited for studying molecules adsorbed at interfaces. VSFG is a second-order nonlinear spectroscopy, which provides us vibrational spectra reflecting molecular structure with high surface specificity.5 So far, many studies of proteins at various interfaces have been performed by using homodyne-detected VSFG, and molecular level insight has been obtained.6−12 Recently, VSFG has been extended to the heterodyne detection scheme, which enables us to determine the phase of © 2015 American Chemical Society

Received: February 27, 2015 Revised: April 2, 2015 Published: April 22, 2015 9947

DOI: 10.1021/acs.jpcc.5b01937 J. Phys. Chem. C 2015, 119, 9947−9954

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The Journal of Physical Chemistry C phase.27 It has been well established that VCD signals of the amide modes sharply reflect molecular conformation of proteins.28 Although VCD is a powerful means of studying the molecular structure of chiral molecules, it is virtually impossible to detect chirality of monolayers by VCD owing to its low sensitivity. Chiral VSFG is promising as a chiral spectroscopic tool, in particular in the application to interfaces. It has been applied to biologically relevant systems such as peptides and proteins at interfaces.29,30 Yan et al. showed that chiral VSFG signals from proteins and peptides were sensitive to their secondary structures.30−32 Recently, we have developed heterodyne-detected chiral vibrational sum frequency generation spectroscopy.41 It enables us to obtain information on molecular chirality, that is, left or right handedness. The measured phase of HD-chiral VSFG signal also provided direct information on whether the signal originates at the interface or in the bulk of the sample. Moreover, the heterodyne-detection scheme improves the signal-to-noise ratio by amplifying weak chiral VSFG signals with a local oscillator. In this study, we report both achiral and chiral HD-VSFG measurements of proteins adsorbed at air/solution interfaces. We examined four proteins, bovine serum albumin (BSA), pepsin, concanavalin A, and α-chymotrypsin, as model systems, the bulk properties of which have been well studied by other methods including X-ray diffraction and vibrational spectroscopy. Table 1 summarizes the content of secondary structures

HD VSFG Setup. Our HD VSFG spectrometer based on a femtosecond laser system is described elsewhere.41 Briefly, a femtosecond regenerative amplifier (Legend Elite, Coherent) was used to generate fundamental pulses (repetition rate 1 kHz, center wavelength 800 nm, pulse width ∼100 fs, average power 3.5 W). One-third of the output was used to pump an optical parametric amplifier (TOPAS-C, Coherent) to generate a broadband infrared beam. The remaining two-thirds was introduced into a narrow band second harmonics generator (SHBC, Coherent). The output was then introduced into an optical parametric amplifier (TOPAS 400, Coherent). The wavelength of the visible beam was set to 630 nm. The infrared and visible beams were overlapped in a y-cut quartz thin plate, whose thickness was 10 μm, to generate a broadband sum frequency generation signal as a local oscillator (LO). The transmitted infrared and visible beams and the LO were refocused onto the sample by a concave mirror. The incident angles of the IR and visible were ∼60° and ∼70°, respectively. The LO passed through a fused silica plate (1.5 mm thickness) between the sample and the concave mirror to delay the LO in time (∼2.5 ps). The reflected LO and the SFG from the sample in reflection passed through an analyzer to select their polarization. They were introduced into a polychromator (TRIAX550, Horiba Jovin Yvon) after a prism monochromator (CT25-UV, JASCO) and interfered with each other in the frequency domain. The interference fringe pattern was finally detected with a liquid-nitrogen-cooled CCD detector (LN/ CCD-1340/400-EB/1, Roper Scientific). The energy of the visible probe at the sample was ∼20 μJ/pulse. That of the IR probe was 8 and 4 μJ/pulse in the CH−OH (NH) stretching region and amide I region, respectively. The SSP and PSP polarization combinations were employed for achiral and chiral measurements, respectively. The samples were placed in a homemade Teflon trough (67 × 67 × 5 mm3). A left-handed zcut quartz plate was used as a reference. The specific configuration of the z-cut quartz was as described in the previous paper.41 The height of the sample surface was monitored by a displacement sensor (LT8110, Keyence) and was maintained within ±8 μm. The exposure time was 1 or 2 min for the samples and 30 s or 1 min for quartz in the achiral VSFG measurements. The exposure time was 1−5 min for the samples and 1 or 2 min for the quartz in the chiral measurements. Each spectrum in this paper was measured without changing the center frequency of the broadband IR light. Concanavalin A is known to form aggregates under laser irradiation.36 In the measurement of the protein, to avoid the aggregation, the exposure time was limited to 1 min and the focusing point was changed to a different point for each measurement. By referring to the phase of the electric field of the chiral signal, we can attribute the origin of the signal to bulk or interface without any ambiguity. This is because the phase of chiral VSFG signals from bulk is different by 90° from that of signals from the interface in the dipole approximation.42 The VSFG electric fields from the bulk and an interface are (2) ̃ ̃ expressed as Ẽ SFG,bulk = rbulkχbulk E1E2 and Ẽ SFG,interface = (2) ̃ ̃ irinterfaceχinterfaceE1E2, respectively, where r are positive real constants and Ẽ 1 and Ẽ 2 are the electric fields of the visible and infrared lasers. Because our reference was the SFG signal of z-cut quartz, Ẽ SFG,quartz, which originates from the bulk, signals from bulk and interface are normalized in the analysis (2) ̃ ̃ procedure as follows, χ(2) eff,bulk = (ESFG,bulk/ESFG,quartz)χquartz and

Table 1. Secondary Structural Content (%) Reported in the Literature BSA38 pepsin39 concanavalin A40 α-chymotrypsin40

α-helix

β-sheet

β-turn

67 12 2 10

0 46 64 51

10 20 22 22

of these molecules obtained in the literature.33 BSA has been established as the standard for the quantification of proteins by UV−vis absorption spectroscopy. BSA is rich in α-helical structure and has been studied in several environments by homodyne-detected VSFG.7,9 We also measured pepsin, concanavalin A, and α-chymotrypsin,34−37 all of which contain significant amounts of antiparallel β-sheet, to examine the relationship between HD-VSFG signals and secondary structures of proteins adsorbed at the interface. From achiral spectra, we can directly deduce the molecular orientation of proteins at the interface. In addition, information on the orientation of water molecules around proteins is obtained. From chiral spectra, the secondary structures substantially contained in proteins can be inferred. In this study, we focus on both X−H (X = C, N, O) stretching regions and the amide I band. Our results clearly show that the HD-VSFG is a powerful probe of molecular orientation and secondary structure of proteins at interfaces in situ.

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EXPERIMENTAL SECTION Chemicals. Bovine serum albumin, α-chymotrypsin, and pepsin A were purchased from Nacalai Tesque. Concanavalin A was from Sigma-Aldrich. All the proteins were used as received. For the solvent, phosphate buffered saline (PBS) from Santa Cruz Biotechnology was used. The pH of the solutions was set to 6.9−7.0. The concentration of the solutions was 2.0 mg/mL. 9948

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Figure 1. Achiral (SSP) complex χ(2) spectra (red, imaginary part; blue, real part) of BSA (A), pepsin (B), and concanavalin A (C) solutions in the CH and OH stretching region. The intensity scales were normalized with the intensity of z-cut quartz.

Figure 2. Achiral (SSP) complex χ(2) spectra (red, imaginary part; blue, real part) of BSA (A), pepsin (B) and concanavalin A (C) solutions in the amide I region. The intensity scales were normalized with the intensity of z-cut quartz. (2) (2) (2) χeff,interface = (iẼ SFG,interface/Ẽ SFG,quartz)χquartz /Δkz, where χeff,bulk (2) and χeff,interface are effective second-order nonlinear susceptibility −13 mV−143), and of the sample, χ(2) quartz is that of quartz (6.0 × 10 1/Δkz is the coherence length, which was set approximately common for the sample and the reference. In the spectral analysis of this study, we assumed the origin of the achiral and chiral signals to the interface to calculate the complex spectra shown in Figures 1−4. The assumption was justified because the imaginary and real parts of the calculated χ(2) spectra could be reasonably regarded as sums of peak and dispersive shapes, respectively. In a previous work, we found that the chiral signal from limonene comes not from the surface but from bulk limonene.41 On the other hand, we assumed that the chiral signals obtained in this study originated from the interface.

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indicate that the hydrophobic methyl group pointed from the protein molecules toward the air. A portion of methyl groups in the protein molecule probably locate in water with the orientation of H atoms pointing down into bulk, but the number of methyl groups in air is larger than that in water. This result suggests that BSA stabilizes at a different molecular structure or conformation at the air/water interface compared with that in bulk water, where the protein is expected to have a tertiary structure with its methyl group folded toward the inside. As in the case of the methyl groups, the sign of the OH stretching band in the Im[χ(2)] spectrum reflects the molecular orientation of water. A positive band indicates water molecules oriented with their H atoms up toward the air.15,17 From this assignment and the observed sign, we conclude that water molecules around BSA were likely to have the orientation with H atoms pointing up. The net charges of the adsorbed proteins induce the ordering of water molecules around the protein monolayer. The pH of the solution in the experiments, ∼7, was above the isoelectric point (pI = 4.9), such that the net charge of the protein should have been negative. This is consistent with the conclusion that the water molecules had the preferential orientation with their H atoms up toward the air. The same orientations of the methyl group and water molecules at the BSA solution interface were proposed by Chen et al.,7 but we first obtained the direct evidence through the heterodyne detection. The spectral features of PBS solutions of pepsin and concanavalin A were almost the same as those of BSA, suggesting that achiral spectral response in the CH stretching region is insensitive to secondary structures. Negative bands due to the CH3 symmetric stretching and a Fermi resonance and a positive band due to the asymmetric CH3 stretching mode were observed. A broad and positive band originating from the OH stretching mode was also observed from two solutions. These results suggest that the methyl groups of the proteins and OH bonds of water are likely to orient with H

RESULTS AND DISCUSSION

Achiral Im[χ(2)] Spectra in the CH and OH Stretching Regions and Amide I Region. Figure 1A−C shows achiral complex χ(2) spectra of BSA, pepsin, and concanavalin A, respectively, in the SSP polarization combination in the spectral range from 2800 to 3400 cm−1. The spectra are dominated by the CH and OH stretching modes. The NH stretching mode might also contribute to the spectra, but it is difficult to distinguish its contribution from broad features due to OH stretching modes. The Im[χ(2)] spectrum of BSA in Figure 1A shows negative bands at 2870, 2933, and 3063 cm−1, which are assigned to the CH3 symmetric stretching, a Fermi resonance, and aromatic CH stretching mode, respectively, and a positive band at 2968 cm−1 due to the asymmetric CH3 stretching mode. In addition, a broad and positive band around 3300 cm−1 due to the OH stretching vibration was observed. In the heterodyne detection, we can obtain additional information on the molecular orientation from the Im[χ(2)] as mentioned in the Introduction. In the case of the methyl group, the negative sign corresponds to the orientation of H atoms of the group pointing toward air.15 The negative bands 2870 and 2933 cm−1 in this study 9949

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Figure 3. Chiral (PSP) complex χ(2) spectra (red, imaginary part; blue, real part) of BSA (A), pepsin (B), and concanavalin A (C) solutions in the NH stretching region. The intensity scales were normalized with the intensity of z-cut quartz.

Figure 4. Chiral (PSP) complex χ(2) spectra (red, imaginary part; blue, real part) of BSA (A), pepsin (B), and concanavalin A (C) solutions in the amide region. The intensity scales were normalized with the intensity of z-cut quartz.

because of the hydrogen bond between the peptide backbones and water molecules. From the SSP spectra, we concluded as follows. First, the methyl groups of the proteins oriented with H atoms pointing toward the air on average probably due to the hydrophobicity of the group, suggesting that the conformation of the proteins at the air/solution interface is different from that in bulk solution. Second, the water molecules around the proteins were likely to preferentially orient with the H atoms pointing toward the air. This is attributed to the net charges of the adsorbed proteins, which depend on the pH of the solution. Third, for the examined three protein solutions, the amide I bands were positive and had approximately the same peak frequency, indicating that the achiral spectral component in the amide I region is less sensitive to secondary structures of interfacial proteins than the chiral signals shown later. Chiral Im[χ(2)] Spectra in the NH Stretching Region and Amide I Region. Figures 3 and 4 show chiral complex χ(2) spectra of BSA, pepsin, and concanavalin A in the NH stretching and amide I regions, respectively. It is worth noting that both imaginary and real parts far from the vibrational resonance were close to zero. This finding is quite a contrast to the fact that the achiral real parts displayed substantial negative signals in the off-resonant region as clearly seen in Figure 2. It was shown in a previous study that an electric quadrupole contribution is significant in the nonresonant background of achiral SFG at interfaces.50 The reason for the absence of the nonrensonant background in chiral spectra is currently unknown. As explained in the Experimental Section, when we normalized spectra of samples with the spectra of the z-cut quartz, we assumed that the origin of the chiral signals was not from the bulk but from the interface. Because Im[χ(2)] in Figures 3 and 4 showed a typical vibrational resonant feature (peak shapes), this assumption was verified. This result is the first piece of direct evidence that interfacial proteins generate

atoms up toward the air. The intensities of the OH stretching modes were slightly different among the three proteins, which may be attributed to the difference in the net charges of the proteins at the air/solution interface. Indeed, pepsin (pI = 1) gave a stronger OH signal than BSA (pI = 4.9) and concanavalin (pI = 5). We now focus on the amide I region. It is well established that the amide I band around 1650 cm−1 is sensitive to the secondary structures of proteins. Recently, in the VSFG studies, the amide I band has drawn much attention experimentally and theoretically for characterizing protein conformation.44−49 Figure 2A−C shows achiral complex χ(2) spectra of BSA, pepsin, and concanavalin A, respectively, in the SSP polarization combination in the spectral range from 1500 to 1800 cm−1. In the Im[χ(2)] spectra, all three protein solutions showed a positive band around 1650 cm−1 due to the amide I mode, even though the contents of secondary structures of the proteins are significantly different as shown in Table 1. While achiral VSFG spectra with a high frequency resolution may characterize secondary structures like IR and Raman spectroscopy, this result suggests that the achiral amide I was less sensitive to the conformations of the examined proteins than the chiral amide I band with the present frequency resolution as reported.31 One possibility is that the disordered part of protein, whose orientation can be controlled by the hydrophobic interaction at the interface, gave rise to the achiral amide I band. A simple quantum chemistry calculation (see details in Supporting Information) shows that the sign of the achiral amide I band reflects the direction of the transition dipole moment of the amide I mode, which is approximately parallel to the CO bond; the calculated sign of the amide I mode is positive when the transition dipole moment is aligned with the C atom of the CO bond oriented to the air. The observed positive amide I bands suggest that the carbonyl group is likely to orient with the C atoms pointing up toward the air. The orientation should be the most stable at the air/water interface 9950

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chiral vibrational band can be changed when the signal is in resonance with electronic transitions. However, we conducted VSFG measurements in electronic nonresonant conditions because the visible probe wavelength (630 nm) was far from the electronic absorption of proteins, which is located in the UV region. Therefore, the electronic resonance effect need not be considered. Thus, the first and second factors should control the sign of chiral VSFG bands. In the present study, all the proteins were composed of amide acids with the same handedness (L-amino acids), suggesting that the second factor similarly contributes to the sign. The model that precisely explains the chiral VSFG intensity and sign has been not developed, even though some attempts have been made.29,44,45 Sign information obtained in the heterodyne detection is valuable in developing and accessing the model for chiral VSFG signal of proteins. Further accumulation of chiral HD-VSFG data from various proteins is highly desirable. Real-Time Observation of Conformational Change of α-Chymotrypsin at the Interface. Figure 5A,B shows the

chiral VSFG signals and thus chiral VSFG is virtually interface specific. Chiral NH Stretching Band. Yan’s group proposed a correlation between secondary structures of proteins and peptides and chiral VSFG bands in the PSP combination for the amide I and NH stretching regions as follows: (i) antiparallel β-sheet rich peptide gives rise to the amide I and NH stretching bands in the PSP combination, (ii) α-helix does not generate the amide I band but rather the NH stretching band, and (iii) disordered state does not result in either the amide I or NH stretching band.32 In the following, the observed chiral VSFG spectra will be discussed referring to the proposed correlation. In Figure 3A, the BSA solution shows a small positive band around 3300 cm−1 in the Im[χ(2)] spectrum. From the previous studies of chiral VSFG, this band is assigned to the NH stretching mode of the protein backbones.51,52 The frequency of the band that we observed is approximately the same as in the previous studies, suggesting that the 3300 cm−1 band in our results originated from protein backbones as well. As clearly seen in Figure 3B,C, the solutions of pepsin and concanavalin A also showed positive bands around 3300 cm−1 that we attribute to the NH stretching mode as for BSA. It is noted that the intensities were much stronger than that of BSA. Chiral Amide I Band. Next, chiral amide I bands will be discussed. Figure 4B,C shows chiral complex χ(2) spectra from concanavalin A and pepsin solutions, respectively. We see clear amide I bands in both spectra. The peak positions are around 1630 cm−1, suggesting that this band is due to antiparallel βsheet structure inferred from the FT-IR studies.53 This result is consistent with the previous study, in which antiparallel β-sheet rich peptide gave rise to the amide I band in the PSP combination as well as the NH stretching band. On the other hand, BSA, which is abundant in α-helix (Table 1), also showed a weak but significant amide I band, while Yan et al. suggested that α-helix structure does not give the amide I band. Provided that α-helix structure does not give the amide I band, one explanation of this band is the conformational change of the protein at the interface. Even though BSA is rich in α-helix, it was reported that BSA adsorbed on highly hydrophilic or hydrophilic interface changes into β-sheet structure in part.54 Moreover, it was shown that heat induces the conformational change of BSA from α-helix to β-sheet aggregates in solution.55−57 Considering these experimental facts, we speculate that BSA changes its conformation driven by the thermal instability at the air/water interface, which is strongly hydrophobic. The Signs of the Chiral Bands. In regard to correlation between secondary structures and chiral VSFG, it is interesting to notice that the chiral amide I and NH stretching bands observed from the solutions of pepsin and concanavalin A had the same positive sign. In addition, as will be shown in the next subsection, the same positive signs were obtained from αchymotrypsin. This is surprising because the orientations and conformations of the three proteins may be rather different from one another, although they all contain the β-sheet as the major secondary structure. The following three factors may affect the sign of the chiral VSFG band: (i) the local handedness of α-carbons adjacent to peptide groups, (ii) the secondary structure and its orientation of aligned protein layer,58 and (iii) electronic resonance effect. Among them, we need not care about the electronic resonance effect. The sign, or phase to be accurate, of the

Figure 5. Time dependence of the imaginary part of the chiral amide I band (A) and the NH stretching band (B) of α-chymotrypsin solution during the 1 h measurements. Time-dependence of the signal intensities of the chiral amide I band (C) and the NH stretching band (D). (E) Imaginary spectrum in red was obtained just after the formation of the surface and that in black was obtained 1 h after the surface formation.

time-lapse observation of the chiral amide I band (1630 cm−1) and NH stretching mode (3300 cm−1), respectively, of αchymotrypsin. The combination of these two bands suggests that the protein is rich in antiparallel β-sheet structure. The measurements started several minutes after the solution was poured into the trough and a stable, flat surface was formed. The spectra were measured every 5 min. Clear bands were observed early in the measurements in both spectral regions, and then the two bands decreased with time, indicating that the 9951

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The Journal of Physical Chemistry C portion of antiparallel β-sheet structure was denaturing. The time dependence of the signal intensities of the chiral amide I and NH stretching bands (Figure 5C,D) indicates that the process was completed about 40 min after starting the measurement. It was confirmed that this denaturation took place at the surface but not in the bulk because mixing of the sample solution recovered the chiral amide NH bands to their original positions and intensities. Achiral spectra also showed a significant change. Figure 5E shows the achiral spectra measured in the CH and OH stretching region just after the formation of the surface and 1 h after that, respectively. The spectrum obtained at an early time (shown in black) in Figure 5E shows almost the same spectral features as Figure 1A−C obtained from the other protein solutions, that is, BSA, pepsin, and concanavalin A: negative bands at 2870, 2933, and 3063 cm−1, due to the CH3 symmetric stretching, a Fermi resonance, and aromatic CH stretching mode, respectively, and a positive band at 2968 cm−1 due to the asymmetric CH3 stretching mode. On the other hand, in the spectrum obtained 1 h later (shown in red), no aromatic band around 3060 cm−1 was observed, the OH stretching band decreased, and a band at 2830 cm−1 newly appeared. The disappearance of the aromatic band and the change in the CH stretching region suggests conformational transformation of the protein at the interface. The decrease of the OH stretching band implies that the randomization of the molecular orientation of water around the protein was associated with the protein deformation. We can summarize the dynamics as follows. First, αchymotrypsin molecules are adsorbed onto the air/water interface within our time resolution of 5 min. At this time, the protein is rich in β-sheet structure as is in the bulk. Next, the adsorbed molecules change their conformation to a disordered state perhaps due to the thermal instability at the interface. This gives rise to the decrease of the chiral amide I and NH stretching bands, which originate from the antiparallel structure. This also changes the achiral spectra in the CH stretching region. Reorientation of water molecules around the protein may play a role in the protein deformation or the protein deformation results in the randomization of water molecular orientation. It should be noticed that proteins remained at the interface, which was confirmed by the achiral measurement, and changed their conformation. We believe that it is difficult to observe the dynamics observed in this study by the homodyne detection due to its low sensitivity and that the improvement of the signal-to-noise ratio by using the heterodyne detection was crucial in the observation of the time-resolved chiral VSFG with the time resolution of 5 min.

indicates the conformational change of the protein at the air/ water interface, which also induced substantial spectral change in the achiral component. Further research on determination of accurate peak positions and band amplitude by HD-chiral VSFG is promising for characterization and quantification of interfacial proteins.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Fitting parameters for χ(2) spectra in Figures 2, 3, and 4 and details of quantum chemical calculations to estimate the SFG molecular hyperpolarizability. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Funding

This study is partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education Culture, Sports, Science, and Technology of Japan (Grant No. 24350010; No. 26104504, Innovative Areas 2503) and by a Grant for Basic Science Research Projects from the Sumitomo Foundation. Notes

The authors declare no competing financial interest.

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REFERENCES

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CONCLUSION In conclusion, we demonstrated the heterodyne-detected achiral and chiral VSFG study of proteins at the air/water interface. We measured four representative proteins, BSA, pepsin, concanavalin A, and α-chymotrypsin in the PBS solution. The orientations of the methyl groups and amide carbonyl groups of the proteins and of water molecules were deduced from the achiral spectra. However, the achiral spectra were similar to one another among the examined proteins. Contrastingly, the chiral spectra in the amide I and NH stretching regions differed among the proteins with different secondary structures. Time-lapse observation of the chiral component of the α-chymotrypsin solution was also performed. The decrease of the chiral amide I and NH stretching bands 9952

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