Broad-Bandwidth Chiral Sum Frequency Generation Spectroscopy for

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Broad Bandwidth Chiral Sum Frequency Generation Spectroscopy for Probing Kinetics of Proteins at Interfaces Zhuguang Wang,1 Li Fu,2 Gang Ma,3 Elsa C. Y. Yan* 1

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,1

Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06520

William R. Wiley Environment Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352

3

Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China

Abstract Kinetics of proteins at interfaces plays an important role in biological functions and inspires solutions to fundamental problems in biomedical sciences and engineering. Nonetheless, due to the lack of surface-specific and structural-sensitive biophysical techniques, it still remains challenging to probe protein kinetics in situ and in real time without the use of spectroscopic labels at interfaces. Broad bandwidth chiral sum frequency generation (SFG) spectroscopy has been recently developed for protein kinetic studies at interfaces by tracking chiral vibrational signals of proteins. In this article, we review our recent progress in kinetic studies of proteins at interfaces using broad bandwidth chiral SFG spectroscopy. We illustrate the use of chiral SFG signals of protein side-chains in the C-H stretch region to monitor selfassembly processes of proteins at interfaces. We also present the use of chiral SFG signals from the protein backbone in the N-H stretch region to probe the real-time kinetics of proton exchange between protein and water at interfaces. In addition, we demonstrate the applications of spectral features of chiral SFG that are typical of protein secondary structures in both the amide I and the N-H stretch regions for monitoring kinetics of aggregation of amyloid proteins at membrane surfaces. These studies exhibit the power of broad bandwidth chiral SFG to study protein kinetics at interfaces and the promises of this technique in the research areas of surface science to address fundamental problems in biomedical and material sciences. Keywords: Protein, Peptide, Kinetics, Vibrational Sum Frequency Generation, Secondary Structures, Broad Bandwidth, Interface

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1. Introduction Protein kinetics at interfaces plays an important role in biology and biomedical sciences and engineering. For instance, life processes rely heavily on the dynamic interactions between proteins and cell membrane surfaces, such as molecular transportation,1 cell adhesion,2 and signal transduction.3 In addition, kinetics of protein aggregation at membrane surfaces is associated with the progression of neurodegenerative diseases.4 Furthermore, proteins, used as therapeutics, can be denatured during storage and manufacturing upon interactions with surfaces of piping and receptacles,5 thus understanding how fast the interaction alters protein structures is crucial for the optimization of shelf life. While proteins and peptides can self-assemble into well-defined structures at various interfaces, the rate of the self-assembly process dictates the stability and synthetic method for manufacturing functional biomimetic materials.6 Moreover, enzymes have been developed into biocatalysts for the transformation of organic compounds and for biofuel cells,7 and their functions and activities are highly correlated to their real-time conformations at interfaces. Hence, in situ and real-time methods for monitoring the kinetics of protein conformational changes at interfaces are crucial in many research fields across various disciplines in surface science. The requirement for the selectivity to molecules at interfaces without interference from bulk media or the use of spectroscopic labels renders it challenging to probe protein kinetics at interfaces. Among the available biophysical techniques, fluorescence spectroscopy often requires labeling,8 potentially perturbing the behaviors of proteins. Nuclear magnetic resonance (NMR) spectroscopy,9 circular dichroism (CD) spectroscopy,10 and dynamic light scattering (DLS)11 are all excellent techniques to probe protein kinetics in bulk solutions, but are unable to obtain structural information at interfaces. Atomic force microscopy (AFM)12 and thin-film X-ray diffraction13 can reveal morphology of protein layers at interfaces, but fall short of molecular information about chemical structures. Although conventional vibrational spectroscopy techniques, such as infrared reflection absorption spectroscopy (IRRAS)14 and surface enhanced Raman spectroscopy15 are able to probe protein kinetics in real time, the

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overlap of characteristic peaks from α-helical and disordered secondary structures in the amide I region (both centered at ~1654 cm-1)16, 17 poses challenges on quantitative interpretation of kinetic data, especially for complex native proteins. Hence, a complementary method is desired to carry out kinetic studies of proteins at interfaces. Chiral sum frequency generation (SFG) spectroscopy is a non-invasive and label-free technique that is able to provide surface-specific and structural-sensitive information about proteins in situ and in real time. Previously, several groups have conducted in situ chiral SFG studies on proteins at interfaces using scanning SFG spectrometer.18-21 However, the acquisition of SFG spectra requires scanning of vibrational frequencies, making real-time kinetic studies difficult. Since the setup of a broad bandwidth SFG spectrometer in our lab in 2008, we have been implementing the chiral SFG method for probing the kinetics of protein structures at interfaces.22-26 The broad bandwidth spectrometer has allowed us to obtain SFG spectra shot-by-shot without spectral scanning,27 thereby advancing SFG spectroscopy into a realtime method for monitoring protein kinetics at interfaces for a time scale of 1-10 minutes.22-25 With the surface specificity and structural selectivity, chiral SFG is able to characterize different secondary structures that are difficult to distinguish using conventional vibrational methods, such as α-helices and disordered structures,22 allowing kinetic studies of protein conformational changes exclusively at interfaces both in situ and in real time.22, 23 Moreover, its chirality-selective characteristic makes chiral SFG a background-free technique since it eliminates the O-H stretch background of water and allow the use of the peptide amide N-H stretch as a new vibrational tool to study protein structures.22, 24 Furthermore, chiral SFG is silent to achiral molecular structures, thus make possible the use of chiral C-H stretch for protein characterization without interference from lipid molecules and other organic compound at interfaces.25 Given the higher laser power used in the C-H stretch vibrational regions, the spectral qualities can be improved and therefore a higher temporal resolution for kinetic studies can be achieved.25 Altogether, the above developments have made broad bandwidth chiral SFG spectroscopy a promising complementary technique for studying kinetics of protein conformational changes at interfaces.

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In this article, we will review the recent progress on the kinetic studies of proteins performed at interfaces. Following a brief introduction of chiral SFG spectroscopy, we will first discuss the development of broad bandwidth SFG spectrometers, which makes it possible to acquire spectra in real time, vital for performing kinetic studies. Then, we will discuss our kinetic studies of proteins using chiral SFG in three vibrational regions: (a) C-H stretch, which can reveal structures of side chains in proteins, (b) N-H stretch, which can be used to monitor proton exchange in the protein backbone, and (c) the amide I region, which can be used to distinguish secondary structures of proteins at interfaces. We will then discuss the outlook of chiral SFG spectroscopy in terms of method developments and potential applications in the broader field of surface science. 2. Basic principles of chiral SFG SFG Method. SFG is a second-order spectroscopic technique pioneered by Shen’s group.28 Rigorous treatment of the SFG theory has been described in excellent books.29, 30 In this section, we focus on vibrational SFG that uses two pulsed laser sources, one at IR frequency ωIR and the other at visible frequency ωVIS (Scheme 1A). When these two beams spatially and temporally overlap at an interface, a new laser beam with the sum of the incident frequencies (ωIR + ωVis) is generated. When the IR frequency (ωIR) is in resonance with molecular vibrations, the SFG signal is enhanced, thereby providing vibrational information about molecules. Since the development of chiral SFG, there have been discussions about whether chiral SFG is surface-specific. We recently discussed in a review article, from the first principle, how to consider the chiral SFG contributions from the bulk versus interface.26 Our theoretical analysis concluded that when an anisotropic interface is situated between two isotropic bulk media under the conditions that electronic resonance is absent, chiral vibrational SFG is surface-specific.26 More specifically, using the dipole approximation, the 2nd-order polarization, P(2), can be induced at the sum frequency (ωIR + ωVis) to coherently generate the SFG electric field:

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 I I (2) J K P (2) = P x xˆ + P y yˆ + P z zˆ ; P ∝ ESFG ∝ ∑ χ IJK EVIS EIR

(1),

JK

where xˆ ,

yˆ , and zˆ are unit vectors; I, J, K (= x, y, or z, laboratory coordinates) specify the vector

direction; χ(2) is a 2nd-order susceptibility tensor element; and EVIS, EIR , and ESFG are the optical fields. IJK The 2nd-order susceptibility of an interface contains a non-resonant term,

, which is independent of IR χ(2) NR

€

frequencies, and a sum of resonant terms, χ(2) , q

χ = χ + ∑χ = χ + ∑ (2)

(2) NR

(2) q

q

€

(2) NR

q

€

Aq

(2),

ω IR −€ω q + iΓq

where Aq, Γq, and ωIR are the amplitude, damping coefficient, and incident IR frequency, respectively; ωq is the vibrational frequency of the qth vibrational mode. As seen in Eq. 2, when the IR frequency (ωIR) coincides with the vibrational frequency (ωq), χ(2) is maximized, yielding amplified SFG electric field according to Eq. 1.

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Scheme 1. (A) Illustration of vibrational sum frequency generation. (B) Illustrations of chiral (C∞) and achiral (C∞V) surfaces. The blue susceptibility elements are non-zero at both chiral and achiral surfaces, and the red ones are non-zero only at chiral surfaces. (C) Illustrations of polarization settings for achiral and chiral SFG experiments: the projection of the electric field of p-polarized and s-polarized light onto the laboratory coordinates. (left) ssp (achiral) polarization setting. (middle) psp (chiral) polarization setting. (right) The definition of polarization.

Chiral SFG. Here, we discuss how chiral SFG differs from achiral SFG. As discussed by Simpson and coworkers,31, 32 an achiral surface adopts C∞V symmetry, leaving seven χIJK elements nonzero (χxxz

= χyyz, χxzx = χyzy, χzxx = χzyy, and χzzz), while a chiral surface adopts C∞ symmetry (Scheme

1B), adding six extra χIJK non-vanishing elements (χxyz,

χyxz, χzxy, χzyx, χxzy, and χyzx). When I≠J≠K, the

2nd-order susceptibility elements exhibit the characteristics of a chiral Cartesian coordinate system, where the three axes x, y, and z are orthogonal to one another. The orthogonal χIJK (I≠J≠K) elements are representations of a chiral surface, which can be measured by using specific polarization settings in an SFG experiment.

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SFG experiments are often performed using linearly s- or p-polarized IR and visible beams, while measuring s- or p-polarized SFG signals. While chiral SFG signals can be indirectly measured with the interference method using a mixed polarization of s and p,33 we used the psp polarization setting (ppolarized SFG, s-polarized visible, and p-polarized IR) to directly observe chiral SFG signals (Scheme 1C, middle). We also used the ssp polarization setting (s-polarized SFG, s-polarized visible, and p-polarized IR) to observe achiral SFG signals (Scheme 1C, left). The SFG intensity measured using these settings can be expressed as, 2

2

psp ssp (2) and I SFG I SFG ∝ χ (2) ∝ χ ssp I vis I IR psp I vis I IR

(3),

(2) (2) where ISFG, Ivis, and IIR are the intensities, and χ ssp and χ psp are the effective 2nd-order susceptibilities,

which can be expressed as linear combinations of χ(2) of the interface (Scheme 1C, left and middle).26 IJK

€

(4),

where αi is the incident/reflected angle of the ith laser beam and L(ωi) is the Fresnel factor. Scheme 1C shows that the χ (2) psp susceptibility is related to orthogonal tensor elements of

χxyz and χzyx, which are

non-zero for a chiral interface (C∞ symmetry). Under the condition of no electronic resonance,

χxyz = 0,

and the psp polarization setting only measures χzyx, which contains structural information about a chiral interface.

3. Broad-bandwidth SFG for studies of protein kinetics at interfaces. Two types of spectrometers, scanning and broad-bandwidth spectrometers, have been used in SFG experiments. The IR bandwidth of the two systems can be determined by the uncertainty principle,

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Δv ∝

1 , where Δt is the pulse width and Δv is the bandwidth. In a scanning spectrometer, picoΔt

/nano-second pulsed laser sources are used to generate both IR and visible beams with a narrow bandwidth. For example, the bandwidth of a 2-ps IR pulse is 10-15 cm-1. Spectra are thus acquired stepwise by scanning the IR frequencies across a vibrational region, such as amide I (1600-1700 cm-1) and C-H stretch (2800-3000 cm-1). In a broad bandwidth spectrometer, a femtosecond IR beam and a picosecond visible beam are used. As an example, our broad bandwidth spectrometer (Scheme 2)27 uses a 100-fs mid-IR laser pulsed beam spanning a bandwidth of 200-300 cm-1, which enables shot-by-shot acquisitions of spectra in a specific vibrational region without scanning the IR frequencies. In the meantime, the bandwidth of the visible beam is narrowed by a pulse shaper to ~10 cm-1, corresponding to pico-second pulses, enabling high spectral resolution. The combination of femtosecond IR and picosecond visible beams reduces the acquisition time of full spectra, and thus facilitates the studies of kinetic processes at interfaces.

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Scheme 2. Illustration of a broad bandwidth SFG Spectrometer. The spectrometer comprises a 6-W

regenerative amplifier (120 fs) seeded by a Ti:sapphire oscillator and pumped by two Nd: YLF lasers. One half of the 6-W output passes through a home-built pulse shaper to produce narrow bandwidth 800nm pulses, and the other half pumps an automated optical parametric amplifier (OPA) to generate broadbandwidth IR pulses. The power of the 800-nm beam at the sample stage is ~135 mW, and the power of the IR beam is ~10 mW. The repetition rate is 5 kHz. The incident angles for the 800-nm and IR beams are 56° and 69°, respectively. The SFG signal generated from the sample is filtered and then dispersed by a monochromator before being detected by a CCD.

The broad bandwidth SFG (BBSFG) spectrometer was first developed by Stephenson’s group.34 Since then, broad bandwidth SFG has been used as a tool for characterization of molecular kinetics at interfaces. Allen’s group compared the bandwidths that can be covered by 80 fs and 1.6 ps IR pulses (Figure 1A),35 and demonstrated the possibility to acquire broad bandwidth IR profiles in the frequency range of 1000-3500 cm-1,36 rendering it possible to obtain high-quality spectra with spectral resolution of < 5 cm-1.37 They obtained full spectra of surfactants at aqueous interfaces with short acquisition time (500 ms) in the C-H region.36 Our group extended the frequency range of broad bandwidths IR to 900-3800 cm-1 (Figure 1B).27 Borguet’s group compressed the IR pulse to ~20 fs and achieved an ultra-broad bandwidth of IR profiles that cover over 1000 cm-1 (Figure 1C).38 Benderskii’s group incorporated heterodyne detection to accurately subtract the non-resonant SFG background and to further improve signal-to-noise ratio (Figure 1D1-D3),39 obtaining phase information to extract molecular-level information, such as absolute orientation.39 Bonn’s group and Tahara’s group40 further applied

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heterodyne detection to the studies of molecular orientation and dynamics at interfaces. Recently, Wang’s group developed a broad bandwidth spectrometer with a spectral resolution of sub-wavenumber (< 1 cm-1) (Figure 1E),41 demonstrating the potential of broad bandwidth spectrometers to probe kinetics of ultrafine structural changes at interfaces. All the recent developments of broad bandwidth SFG spectrometers have widened the applications of SFG and can potentially be implemented to kinetic studies of proteins at interfaces to improve spectral and temporal resolutions.

Figure 1. (A) Comparison of 80 fs and 1.6 ps IR profile bandwidths. Reprinted with permission from

Reference 35. Copyright 2001 the Japan Society for Analytical Chemistry. (B) IR profiles of a typical femtosecond BBSFG spectrometer in the mid-IR 900-3800-cm-1 region. Reprinted with permission from Reference 27. Copyright 2009 Springer. (C) IR profiles of an ultra-BBSFG spectrometer. Reprinted with permission from Reference 38. Copyright 2011 Optical Society of America. (D1) Homodyne-detected and (D2) heterodyne-detected SFG C-H stretch spectra of saturated octanol/d-octanol monolayers (octanol mol fraction indicated) at different surface coverages obtained using a 100 s acquisition time, with (D3) enlargement of the heterodyne-detected spectrum of the 6% monolayer. (D1-3) Reprinted with permission from Reference 39. Copyright 2008 American Chemical Society. (E) Spectrum of the 800 nm visible picosecond (ps) pulse with FWHM of 0.55 ± 0.01 cm−1. Reprinted with permission from Reference 41. Copyright 2013 AIP Publishing LLC.

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4. Kinetic studies on proteins at interfaces using chiral broad bandwidth SFG In this section, we will discuss the applications of broad bandwidth chiral SFG for probing kinetics of (1) protein assembly process using the chiral signal of C-H stretch,25 (2) H/D exchange using the chiral signal of N-H stretch,24 and (3) early-stage amyloidogenesis using the chiral signals of amide I and N-H stretch.22, 23 4.1 Chiral C-H stretch for probing kinetics of self-assembly of proteins at the interface Self-assembly of biomacromolecules at interfaces is important in many research fields, such as polymer sciences, material sciences, and supramolecular chemistry.42 To monitor the self-assembly processes of proteins or peptides, the amide I vibrational signal is often used.16, 17 However, the amide I vibration does not reveal structural information about side-chains, hampering the comprehensive understanding of self-assembly mechanisms. In addition, many biomacromolecules, such as DNA, RNA, and synthetic biopolymers do not have amide groups. Given the abundance of hydrocarbyl groups in various chiral biomacromolecules, it is useful to establish C-H stretch vibrations as a probe for the selfassembly process of chiral molecules at interfaces using chiral SFG spectroscopy. Here, we describe our studies of real-time kinetics of the LK7β peptide at the air/water interface using the chiral C-H stretch signals from the leucine side chains.25 The LK7β peptide is an amphiphilic peptide with a sequence of LKLKLKL. It forms anti-parallel β-sheets at the air/water interface at neutral pH,43 as evidenced by the characteristic ~1619 cm-1 (B2 mode) and ~1680 cm-1 (B1 mode) peaks in the amide I region (Figure 2).24 Using chiral SFG, we observed in real time the self-assembly of LK7β at the air/water interface.25 We combined these kinetic results with the surface pressure measurement and proposed a molecular mechanism of the self-assembly process for LK7β at the air/water interface, as described below.25

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Figure 2. Chiral amide I spectra of LK7β at the air/H2O interface (blue) and air/D2O interface (red) at pH

7.4. Reprinted with permission from Reference 24. Copyright 2013 American Chemical Society.

In the kinetic studies, we first acquired the in situ chiral SFG spectra of LK7β (~25 µM concentration) at the air/water interface at different pH values of the bulk solution, using an acquisition time of 1 min (Figure 3).25 At pH ~7.4, LK7β exhibits strong chiral C-H stretch signals, confirming the formation of chiral secondary structures at the interface. Figure 3 shows that the chiral C-H signal intensity plummets to a negligible level after addition of HCl to bring the bulk pH down to ~1.2, which is due to the denaturation of ordered chiral structures into disordered achiral structures. Then, Figure 3 also shows the recovery of chiral SFG signal ~3 h after addition of NaOH solution to bring the pH back to ~7.4. The recovered intensity level is slightly lower than that of the initial state. The lower intensity is likely due to the dilution of LK7β bulk concentration from pH adjustment, which leads to lower surface density at the interface in equilibrium. The recovery of the chiral C-H signals indicates the reassembly of LK7β at the interface.

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Figure 3. In situ chiral SFG spectra of LK7β at the air−water interface in the C-H stretch region. Spectral

deconvolution is provided for the assembled and the re-assembled states. The insets show the spectral fitting in the region from 2850 to 2920 cm−1. Reprinted with permission from Reference 25. Copyright 2013 American Chemical Society.

Hence, we could use the chiral C-H stretch signals to monitor the kinetics of the self-assembly process of LK7β at the air/water interface in real time (Figure 4A).25 At time = 0 min, we added NaOH to bring the pH back to ~7.4. The signal remains almost silent for the first 60 min, followed by the steady increase during the next 70 min. The signal holds at the maximum level for at least 30 min before we ended the experiment. To confirm the kinetics, we repeated the experiments for four additional times and monitored the intensity of the highest peak (2958 cm-1) in the spectra (Figure 4B), where the red curve corresponds to the result in Figure 4A. While the time taken for the recovery of chiral SFG signal in the 5 experiments ranges from ~70 min to ~137 min, it is still on the same order of magnitude. Most importantly, the kinetic curves reveal a lag phase before the buildup of chiral SFG intensities during the self-assembly of LK7β into chiral structures at the interface. To explore the molecular mechanism for the lag phase, we conducted surface pressure measurements of the self-assembly process (Figure 4C).25 We first set the surface pressure of water to zero. Once LK7β is added to the buffer solution at pH ~7.4, the surface pressure immediately rises. Because surface pressure is positively correlated with molecular surface density, this observation

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indicates a fast adsorption of LK7β at the interface. Upon the change of pH to ~1.2, the surface pressure drops due to desorption (Figure 4C). After pH neutralization to ~7.4, surface pressure gradually increases and reaches a plateau at ~76 min, followed by another increase until attaining equilibrium. Combining the results of chiral SFG studies in the C-H stretch region and surface pressure measurements, we proposed a two-stage self-assembly mechanism for LK7β at the air/water interface (Figure 4D).25 Upon neutralization of pH, LK7β starts to adsorb at the interface, which increases the surface pressure rapidly until reaching a plateau. In this stage, LK7β is largely disordered, thereby exhibiting no chiral C-H SFG signal. At ~1 h after neutralization of pH, LK7β in disordered structures continues to populate the interface and reach a critical point that triggers the self-assembly of disordered structures into anti-parallel β-sheet structures. This is energetically favorable because the folded antiparallel β-sheet structures are more compact, which allow further adsorption of the amphiphilic LK7β peptide onto the air/water interface. Correspondingly, the increases in both the chiral C-H stretch signals and the surface pressure at ~1 h after pH neutralization are attributed to the simultaneous adsorption and self-assembly of LK7β into anti-parallel β-sheet structures at the interface (Figure 4D).

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Figure 4. (A) Time-dependent cSFG spectra showing the self-assembly of LK7β at the interface in C−H

stretch region. At t = 0, NaOH is added to change the pH back to ∼7.4. (B). Normalized intensity of the CH3 AS peak (2958 cm−1) as a function of time during the self-assembly process of LK7β at the air−water interface. The experiment is repeated for five times. (C) Surface pressure measurement for the denaturation and re-assembly of LK7β at the air/water interface. (D) Illustration of LK7β self-assembly mechanism at the air/water interface. (A)-(C) reprinted with permission from Reference 25. Copyright 2013 American Chemical Society.

To underscore the advantages of chiral SFG to probe the kinetics of chiral macromolecular structural changes at the interface, we also used achiral SFG to repeat the above experiments. The in situ achiral SFG spectra do not exhibit consistent peak shapes and relative peak intensities for the assembled states (Figure 5A1 and A3) at different probing spots of the air/water interface in the beaker containing the sample (a, b, c in Figure 5A1 and A3).25 Moreover, prominent achiral SFG signals can be observed when LK7β is in disordered structures at pH 1.2 (Figure 5A2).25 As a result, the time-dependent achiral spectra do not show an obvious trend of changes in intensity during the self-assembly process upon pH neutralization. Consequently, the results of the kinetic experiments using achiral SFG are not reproducible (Figure 5C1-2).25

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Here, we offered the simplest explanation for the inconsistency of achiral spectra. We attributed the irreproducible achiral SFG spectra to the inhomogeneity at the interface in terms of achiral structures. When the self-assembly reaches equilibrium, there can still be a small proportion of peptide molecules that does not form anti-parallel β-sheets. They can exist as either disordered or partially assembled intermediate structures, all of which can contribute to the achiral spectra. The partially assembled intermediates can be largely inhomogeneous in terms of structures and orientations that complicate the achiral spectra. On the other hand, only assembled chiral anti-parallel β-sheets can contribute to the chiral spectra. Since the assembled β-sheet structures with the leucine residue pointing to the air phase and the lysine residue pointing to the water phase, the assembled chiral structures are relatively homogeneous. Thus, chiral spectra are more reproducible at different probing spots than the achiral ones. Based on this interpretation, both achiral and chiral spectra should be reproducible at different probing spots when the interface is covered with only one kind of structure. Indeed, this is what we observed at acidic pH 1.2, where the interface is relatively homogeneous with the coverage of completely disordered structures (Figure 5A2).

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Figure 5. Achiral and chiral SFG spectra characteristic of assembled, denatured, and re-assembled states

of LK7β at the air/water interfaces. (A1-3) Achiral in situ spectra. (B1-3) Chiral in situ spectra. The letters a, b, and c in the figure represent diﬀerent probing areas at the interface, and the same letter in ssp and psp spectra represents the same area. The highest peak intensities are scaled to the same value for the convenience of comparison. (C1-2) Time-dependent achiral (ssp) C−H stretch spectra showing the reassembly of LK7β at the interface in two independent experiments. At t = 0, NaOH was added and the pH was brought back to ∼7.4 to trigger the self-assembly process. Reprinted with permission from Reference 25. Copyright 2013 American Chemical Society.

This work exhibits the ability of chiral C-H stretch SFG signals to reveal the kinetics of selfassembly of peptide at interfaces. This study implies that the chiral C-H SFG signals can also be a promising tool for probing a wide range of macromolecules other than proteins abundant in C-H groups at interfaces, including DNA, RNA, synthetic chiral polymers and macromolecular structures. The study also implies that the chiral C-H stretch SFG signals can be useful in exploring kinetic mechanisms of these molecules at interfaces. The relatively high IR power and no interference from atmospheric water vapor and CO2 in the C-H stretch region contribute to higher signal-to-noise ratio, shortening the acquisition time and thus improving the time resolution for kinetic measurements. The use of chiral C-H stretch signal is expected to advance the applications of chiral SFG to address fundamental and

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engineering problems not only in biological sciences, but also in polymer science, supramolecular chemistry, and material science.42

4.2. Chiral N-H for probing hydrogen-deuterium exchange in proteins at the interface Hydrogen/deuterium (H/D) exchange is a chemical reaction where a covalently-bonded hydrogen atom is replaced by deuterium, or vice versa. The H/D exchange has long been useful for characterization of protein structures and dynamics,44 providing information about protein stability, protein folding, solvent exposure, and conformational changes.45 Various techniques have been developed to probe the H/D exchange in the bulk, including NMR,46 mass spectrometry,47 and FTIR48. However, there is a still a dearth of in-depth studies on protein H/D exchange at the interface, mostly due to technical challenges of acquiring surface selective signals. In addition, because of the close proximity in frequency of the N-H (or N-D) and O-H (or O-D) stretches, the N-H (or N-D) stretch signal from protein backbones is often overwhelmed by the broad O-H (or O-D) stretch vibrational band of water (Figure 6),22, 26 making it impossible to monitor H/D exchange by probing N-H or N-D stretch signal using conventional (achiral) SFG. In contrast, chiral SFG can probe H/D exchange without the interference from the O-H (or O-D) water background because chiral SFG is sensitive to the N-H stretch of protein backbone in chiral secondary structures but insensitive to achiral water structures at interfaces.22, 26 In the study described below, we used chiral N-H/N-D SFG signal to probe the kinetics of H/D exchange of proteins at interfaces.

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Figure 6. Achiral SFG spectra of in the N-H stretch region are usually broad and featureless due to the

interference of O-H stretch in the water molecules. Reprinted with permission from Reference 26. Copyright 2014 American Chemical Society.

In the study, we used LK7β as a model system for H/D exchange study. We first obtained the chiral amide I spectra at the air/H2O and air/D2O interfaces (Figure 2),24 and confirmed the formation of anti-parallel β-sheets with the peaks at ~1619 cm-1 (B2 mode) and ~1690 cm-1 (B1 mode)16, 17. Then, we switched the IR frequency to obtain the chiral SFG spectra in the N-H stretch and the N-D stretch regions (Figure 7).24 In the N-H stretch region, we observed a major peak at 3268 cm-1 and a shoulder at 3178 cm1

. In the N-D stretch region, we observed a major peak at 2410 cm-1 and a shoulder at 2470 cm-1. Based on

normal mode analysis from ab initio computations24 and previous empirical results,49 we assigned the major peaks to the N-H (3268 cm-1) and N-D (2410 cm-1) stretches of the peptide backbone. Here, we eliminate the possibility of contribution from the lysine side-chains in LK7β to the two major peaks, mainly based on the analyses of kinetics, peak positions, and ionic states for –NH3+.24 We also attributed the 3178-cm-1 shoulder to the combination of amide I and amide II vibrations, and proposed the 2470-cm1

shoulder to the combination band of C-N stretch and N-D bending.24

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Figure 7. Chiral SFG spectra of LK7β (A) in the N–H stretch region at the air/H2O interface and (B) in

the N–D stretch region at the air/D2O interface. Reprinted with permission from Reference 24. Copyright 2013 American Chemical Society.

We then monitored the H/D exchange kinetics of LK7β at the air/water interface in real time, which was initiated by adding D2O (or H2O) to the solution of LK7β prepared with H2O (or D2O) (Figure 8A-F).24 Figure 8A-B show a gradual buildup of N-H signal due to the replacement of N-D by N-H. The time for the exchange process is shortened from ~11 min (Figure 8A) to ~7 min (Figure 8B) when the D2O:H2O ratio changes from 4:1 to 2:1. Similarly, the buildup of N-D signal due to the replacement of NH by N-D is also accelerated when the D2O:H2O ratio changes from 4:1 to 2:1. The exchange time decreases from ~53 min (Figure 8C) to ~36 min (Figure 8D). Notably, the rate of exchange from N-D to N-H is faster than that from N-H to N-D for both ratios of 2:1 and 4:1 (Figure 8E-F). The results suggest that the rate-determining step of H/D exchange in LK7β is the breaking of O-H or O-D bond (Figure 8G) with the following argument. Because O-D stretch has lower zero-point energy than O-H stretch, it requires higher energy to break the O-D bond in the exchange of N-H to N-D than to break the O-H bond in the exchange of N-D to N-H. The higher energy barrier therefore leads to a slower rate. This also agrees with previous studies of H/D exchange in crystalline α-cyclodextrin by Ribeiro-Claro’s group, which conclude that the N-D to N-H exchange is 7-10 times faster than the N-H to N-D exchange due to the rate-limiting step of breaking the water O-H or O-D bond.50

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Figure 8. Kinetics of H/D exchange in LK7β at the air/water interface. Time-dependent N−D stretch

spectra of LK7β at the air/H2O interface upon addition of (A) H2O and (B) D2O at a ratio of D2O:H2O equal to 4:1. (C) The time dependence of the SFG field in D-to-H exchange with 4:1 (red) and H-to-D exchange with 4:1 (blue) ratio. (D)-(F) show the results obtained at the ratio of 2:1. (G) Explanation for the difference in H-to-D and D-to-H exchange kinetics. (A)-(F) reprinted with permission from Reference 24. Copyright 2013 American Chemical Society.

This work shows the power of chiral SFG to probe the protein kinetic processes at interfaces hardly accessible to conventional surface characterization techniques. Without the water background in the N-H stretch region, the proton exchange kinetics at the interface can be readily monitored by chiral SFG without isotopic labeling. Kinetic studies of H/D exchange in proteins using chiral SFG also hold promises in revealing molecular mechanisms of various biological processes at interfaces, such as solvent accessibility of proteins in membranes, protein-mediated proton transfer across membranes, and intermolecular and intramolecular hydrogen-bonding interactions in membrane proteins.51

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4.3. Chiral amide I and N-H stretch for probing protein folding and misfolding at the interface As shown in the previous section, chiral SFG can selectively probe the N-H stretch signal from protein backbones, without the interference from water background and side-chains. We further showed that chiral N-H stretch in combination with the chiral amide I signals can be used to characterize protein secondary structures at interface. We took in situ chiral SFG spectra of proteins with known secondary structures at the air/water interface (Figure 9 and Figure 10).22, 26, 52 Table 1 shows a summary of the observations. A comparison of spectra for α-helix and disordered structures highlights the advantage of chiral SFG in distinguishing protein secondary structures. As probed by conventional vibrational spectroscopy, both α-helix and disordered structures exhibit amide I vibrational bands that overlap at ~1654 cm-1. In the applications of chiral SFG, while it is difficult to distinguish the two types of structures by the amide I spectra alone, the prominent chiral N-H stretch peak for α-helices but not for disordered structures can clearly distinguish these two structures (Figure 9 and Table 1).22, 26 Therefore, chiral amide I and N-H stretch SFG signals together can be used as optical signatures to distinguish secondary structures that conventional vibrational spectroscopy may find difficult to differentiate.

Figure 9. Chiral (psp) N-H stretch and amide I SFG spectra for a series of model proteins and peptides

with schematic structures. Reprinted with permission from Reference 26. Copyright 2014 American Chemical Society.

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Figure 10. Chiral (psp) SFG spectra of hIAPP (a model peptide with parallel β-sheet secondary structure)

at different interfaces. (A) Tilted hIAPP yields prominent chiral amide I but no chiral N-H stretch signal. (B) hIAPP lying flat at the interface yields prominent chiral amid I and N-H stretch signals. Color codes: green: hydrophilic residues; White: hydrophobic residues; blue: positively charged arginine residues. Reprinted with permission from Reference 52. Copyright 2015 American Chemical Society.

Table 1. Chiral SFG spectral features in the amide I and the N-H stretch regions for different secondary structures at interfaces summarized from Figure 9 and Figure 10. Model peptide/proteins Tachyplesin I, LK7β Rhodopsin, pHLIP, LK14α Alamethicin rIAPP hIAPP (lipid/water) hIAPP (glass slide)

Secondary structures Anti-parallel β-sheet α-helix 310-helix Disordered Parallel β-sheet (tilted) Parallel β-sheet (lying flat)

Amide I signal Yes No No No Yes (with shoulder at 1660-1670 cm-1)

N-H stretch signal Yes Yes (low cm-1) Yes (high cm-1) No No Yes

We used the chiral SFG vibrational signatures to probe kinetics of amyloid aggregation at interfaces. Amyloid aggregation is associated with various neurodegenerative diseases. Latest clinical studies showed that lipids are reliable biomarkers for detecting amyloid diseases.53 Also, interactions with the membrane can catalyze the aggregation, and small prefibrillar aggregates can perturb the cell membrane and cause cytotoxicity.54 Thus, a detailed molecular understanding of the role of lipid membranes can potentially change the landscape of future approaches for developing treatments.

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Nonetheless, most molecular studies of amyloid aggregation have been performed in the solution phase. Thus, there is a gap of knowledge on the early stages of amyloid aggregation on membrane surfaces. This gap is mostly due to the lack of label-free methods that can detect in situ and in real time protein conformational changes on surfaces without the interference of signals from water and proteins in solution. This knowledge gap can only be bridged by the innovation of technology that is sensitive to both protein structures and interfaces. We used the chiral SFG method to monitor the early-stage aggregation at the air/water interface in the presence of lipid monolayer for human islet amyloid polypeptide (hIAPP), a 37-residue peptide of which the aggregation on membrane surface is associated with the onset of type II diabetes.4 We performed two experiments, in which we took the chiral SFG spectra of hIAPP at the air/water interface in the presence and absence of negatively charged dipalmitoylphosphatidylglycerol (DPPG). In the first experiment, we added hIAPP at the air/water interface and took the chiral SFG spectra in the amide I and N-H stretch regions (right in Figure 11).22 Within 10 hours, the spectra showed no appreciable signal in either region, indicating that hIAPP is disordered. In the second experiment, we initially added the same amount of hIAPP at the air/water interface. Once the adsorption of hIAPP reaches equilibrium, we added DPPG at the interface to form a monolayer and took the chiral amide I and N-H stretch spectra after ~10 h (left, Figure 11).22 The N-H stretch spectrum shows no chiral SFG signal while the amide I spectrum shows a major peak at ~1622 cm-1 and a shoulder at ~1660 cm-1. The two peaks in the amide I region corresponds to amide I A mode (1660 cm-1) and amide I B mode (1622 cm-1) of parallel β-sheets, respectively, indicating the formation of β-sheet-rich hIAPP aggregates at the interface.

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Figure 11. Scheme for the in situ chiral SFG experiments for hIAPP at the air/water interface. Spectra in

the absence of DPPG lipid monolayer (right) and in the presence of DPPG lipid monolayer (left) are shown for the amide I and the N-H stretch regions 10 h after the addition of hIAPP. Reprinted with permission from Reference 22. Copyright 2011 American Chemical Society.

To further examine the pathway through which hIAPP folds into the β-sheet rich aggregates, we monitored in situ and in real time the chiral SFG signal in the amide I and N-H stretch regions. Figure 12A22 shows that after the addition of DPPG to initiate the aggregation, the chiral N-H signal increases and reaches maximum at around 3 h while the chiral amide I signal remains undetectable. After 3 h, the chiral N-H signal fades away but the chiral amide I signal gradually builds up. This transition persists until ~10 h, when the chiral N-H signal completely vanishes and the chiral amide I signal reaches a maximum. To confirm these observations, we repeated the experiments three times and obtained three sets of data exhibiting similar kinetics for the chiral SFG signal in the amide I and N-H stretch regions (Figure 12B).22 Since chiral SFG signal close to 3300 cm-1 is characteristic of α-helix, the observations leads to the conclusion that hIAPP first forms α-helical intermediate, and then aggregates into parallel βsheets (Figure 12C).22 This pathway agrees with a previously proposed working model for hIAPP aggregating on membrane surfaces.55

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Figure 12. Aggregation of hIAPP. (A) The time-dependent chiral SFG spectra in the vibrational regions of

N–H stretch (left) and amide I (right) after addition of DPPG. (B) The intensity of the N–H stretch and amide I signals as a function of time. Results of triplicate experiments are shown. (C) The aggregation model of hIAPP on a membrane surface as observed in the SFG experiments: adsorption as a random coil leads to formation of α-helical intermediates, which are converted to β-sheet aggregates. (D) The timedependent achiral SFG spectra in the amide I region. (A)-(C) reprinted with permission from Reference 22. Copyright 2011 American Chemical Society.

As a control to confirm the conformational changes of proteins at the interface, we also monitored the achiral SFG spectra in the amide I region in real time (Figure 12D).23 Prior to the addition of DPPG, the achiral spectrum shows a peak at ~1650 cm-1, which can be assigned to disordered structures according to chiral experimental results. Upon addition of DPPG to initiate the aggregation, the achiral SFG spectrum shows a C=O stretch peak at ~1740 cm-1 due to DPPG. In the subsequent 10 h, the achiral amide spectrum exhibits a gradual shift of peak position to ~1660 cm-1 accompanied by the slight increase of peak intensity. This observation indicates conformational changes at the interface, supporting the conclusion from chiral SFG experiments. Nevertheless, the 1650 cm-1 achiral SFG peak before the start of aggregation has ambiguous assignments. It can be assigned to either disordered structures or α-helices. Therefore, without spectral information from other vibrational regions, such as the N-H stretch, it is difficult to make definitive conclusion about the secondary structures of proteins during the aggregation of hIAPP using achiral SFG alone.

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This work has illustrated the application of chiral SFG to probe the aggregation kinetics of amyloid proteins at the membrane surface. With its selectivity to chirality, chiral SFG can provide background-free information without the interference from achiral molecules. Due to the silent C=O stretch signal from achiral lipid head groups, characteristic vibrational signals of proteins in the amide I region can be detected without interferences from lipid signals, rendering its application for studying protein aggregation on lipid membrane surfaces. In addition, the absence of the O-H stretch signal from water solvent furnishes the opportunity to use the N-H stretch from protein backbones as an extra handle to track real-time conformational changes of proteins at interfaces. The series of chiral SFG spectra in both the amide I and the N-H stretch region for proteins can serve as vibrational signatures to distinguish secondary structures in proteins. Furthermore, our study also implies the application of chiral SFG in studying the effect of inhibitors or drug candidates on the aggregation of amyloid proteins at membrane surfaces, providing direct information to evaluate drug efficacy and guide rational design of drugs for amyloid diseases. This method can be generally applied to study early stages of misfolding of other amyloidogenic proteins, such as amyloid-β, α-synuclein, and huntingtin, which are associated with neurodegenrative Alzheimer’s, Parkinson’s, and Huntington diseases, respectively.4 Our work also shows the promise of chiral SFG to solve problems related to protein folding in 2-dimenstional surfaces and the functions of intrinsically disordered proteins that changes conformation upon interactions with interfaces.

5. Summary and Outlook To summarize, we have discussed the theoretical background of chiral SFG and the development of broad bandwidth SFG spectrometers for kinetic studies at interfaces. We have also reviewed three applications of chiral SFG spectroscopy on protein kinetics at interfaces: (1) the C-H stretch from protein side chains for monitoring protein assembly processes, (2) the N-H stretch for observing interfacial proton exchange on protein backbones, and (3) the amide I and the N-H stretch for probing the kinetics of the

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early stages of amyloid protein aggregation at the lipid/water interfaces. These kinetic studies have demonstrated the capacity of chiral SFG as a background-free method for studying protein kinetics without the use of spectroscopic labels in situ and in real time at interfaces. The chiral SFG method can be further developed to widen its applications to tackle problems related to more complex molecular systems. In terms of instrumentation, the signal-to-noise ratio is a limiting factor in the temporal resolution of kinetic studies and can be improved by the implementation of several techniques, including heterodyne-detection,39, 40 total internal reflection setup,56 and doublyresonant SFG,57 although the surface-specificity of doubly-resonant SFG remains controversial.26 In terms of data interpretation, various methods have been developed to calculate hyperpolarizabilities for biological macromolecules that allow quantitative spectral interpretation for extracting structural and orientation information.58-60 Moreover, the autocorrelation of second-order optical signals should, in principle, further enable dynamic studies of biomacromolecules at interfaces, as demonstrated by Conboy’s group61 and Eisenthal’s group62. In terms of sample preparation, instead of simple peptides synthesized by solid-state methods, complex proteins can be expressed and purified for SFG studies22 to address biologically relevant problem beyond simple model systems. In addition, isotopic labeling can also be implemented to aid spectral assignments to resolve the ambiguity in interpretations of vibrational spectra for kinetic studies.63 The above developments can help chiral SFG to address a wider range of problems related to the kinetics of proteins at interfaces. For instance, chiral SFG can be used to study protein folding, protein functions, and protein-ligand interactions at interfaces, such as membrane surfaces. The use of peptide backbone N-H/N-D stretch for probing proton exchange can be applied to investigate hydrogen-bonding interactions, solvent accessibility, and protein dynamics at interfaces. Moreover, chiral SFG should be a promising tool to reveal molecular mechanisms of intrinsically disordered proteins that change conformation drastically upon binding to their interacting partners, e.g. various biological interfaces. It can also be used to study amphiphilic proteins, such as constituent proteins in bacterial biofilm

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extracellular matrices for bacterial growth into communities on surfaces.64 Moreover, the use of dynamic chiral SFG signals can potentially inspire research into problems of immobilization of proteins on solid substrates,65 immunological responses of antibodies binding with its antigen,66 and the biocompatibility of implanted medical devices.67 Besides proteins, chiral SFG can also be applied to study kinetics of other important chiral biomacromolecules and synthetic molecular systems. For instance, chiral SFG has revealed the structures of oligonucleotides tethered at interfaces.33 Moreover, characterization of the chiral porphyrin aggregates within the hydrophobic region of lipid bilayers in plant photosynthetic processes has provided insights into the relationship between their structures/orientation and remarkable electronic properties in the light harvesting system.68 By monitoring the structural changes of polysaccharides on plant cell wall matrices, chiral SFG can be used as a potential tool to understand their influences on the formation of cellulose microfibrils.69 Chiral SFG is also a useful tool to probe the self-assembly of chiral polymers/biopolymers/biocolloids at interfaces,70 which provides quality control over the growth of materials through real-time structural characterization. Similar approaches can also be applied to chiral liquids,71 nanoparticles,72 and biomimetic materials73 at interfaces. With a fundamental understanding of interfacial kinetics, chiral SFG can potentially identify new problems and inspire new answers to the questions posed in electronics and engineering, such as the developments of biosensors,74 DNA microarrays,75 biofuel cells,7 semiconductor chips,76 and surface plasmonic techniques77. Hence, further development of the chiral SFG method in conjunction with other characterization methods can introduce opportunities in solving problems to the interest of the greater community of surface science.

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Acknowledgement E.Y. is the recipient of the Starter Grant Award, Spectroscopy Society of Pittsburgh. This work was supported by the National Science Foundation (NSF) Grant CHE 1213362 and the National Institutes of Health (NIH) grant 1R56DK105381-01. The authors thank Ya-Na Chen (Yale University) and Wei Liu (Yale University) for technical assistance.

Zhuguang Wang graduated from the University of Science and Technology of China in 2010 with a B.S. in Chemical Physics. Afterwards, he joined Elsa Yan's research group at Yale Chemistry to carry out his Ph.D. studies. He is currently working on the application of chiral sum frequency generation spectroscopy in the characterization of structures, kinetics, and orientations of biomacromolecules at interfaces. With his research, he aims to reveal molecular mechanisms for biological processes at biomembranes using both experimental and theoretical tools.

Li Fu graduated from the University of Science and Technology of China as an undergraduate in 2008. After earning his B.S. in Chemical Physics, he became a graduate student under the advisory of Elsa Yan at Yale Chemistry, working on the development of chiral vibrational sum frequency generation spectroscopy to investigate biomacromolecules at interfaces. He graduated from the Yan lab in 2013 with a Ph.D., and became a William Wiley Postdoctoral researcher at the Environmental Molecular Sciences Laboratory (EMSL) of Pacific Northwest National Laboratory (PNNL).

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Gang Ma obtained his Ph.D. in chemistry from Peking University in 1999 in the group of Prof. Jinguang Wu. From May 2000 to January 2008, he was a postdoctoral researcher and then research associate in the group of Prof. Heather Allen at The Ohio State University. From February 2008 to January 2010, he was an associate research scientist in the group of Prof. Elsa Yan at Yale University. Gang Ma joined the faculty at Hebei University in 2010 and he is currently a Full professor of Chemistry in the College of Chemistry and Environmental Science.Current research in Prof. Ma’s group focuses on using FTIR spectroscopy and AFM to study protein amyloid formation.

Elsa Yan went to college in the Chinese University of Hong Kong and received her B.Sc. in 1995. She then joined the Eisenthal’s lab to pursue her Ph.D. degree, applying nonlinear spectroscopy to the studies of colloidal interfaces. Following her graduation in 2000, she became a joint postdoctoral fellow with Richard Mathies at UC Berkeley and Thomas Sakmar at the Rockefeller University, where she conducted research on the G-protein coupled receptor rhodopsin with the combined methods of Raman spectroscopy and molecular biology. After working as a Research Assistant Professor at the Rockefeller University, she joined the faculty of Yale Chemistry as an Assistant Professor in 2007 and was promoted to Full Professor in 2014. Elsa’s research centers on applying physical chemistry methods to address molecularlevel biological questions related to biomembranes and membrane proteins from a fundamental perspective.

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References: 1. Borst, P.; Elferink, R. O., Mammalian Abc Transporters in Health and Disease. Annu. Rev. Biochem. 2002, 71, 537-592. 2. Keselowsky, B. G.; Collard, D. M.; García, A. J., Surface Chemistry Modulates Fibronectin Conformation and Directs Integrin Binding and Specificity to Control Cell Adhesion. J. Biomed. Mater. Res. A 2003, 66A, (2), 247-259. 3. Rosenbaum, D. M.; Rasmussen, S. G. F.; Kobilka, B. K., The Structure and Function of GProtein-Coupled Receptors. Nature 2009, 459, (7245), 356-363. 4. Chiti, F.; Dobson, C. M., Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333-366. 5. Dixit, N.; Maloney, K. M.; Kalonia, D. S., Protein-Silicone Oil Interactions: Comparative Effect of Nonionic Surfactants on the Interfacial Behavior of a Fusion Protein. Pharm. Res. 2013, 30, (7), 18481859. 6. Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K., Peptoids: A Modular Approach to Drug Discovery. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, (20), 9367-9371. 7. Sheldon, R. A., Enzyme Immobilization: The Quest for Optimum Performance. Adv. Synth. Catal. 2007, 349, (8-9), 1289-1307. 8. Weiss, S., Fluorescence Spectroscopy of Single Biomolecules. Science 1999, 283, (5408), 16761683. 9. Sattler, M.; Schleucher, J.; Griesinger, C., Heteronuclear Multidimensional Nmr Experiments for the Structure Determination of Proteins in Solution Employing Pulsed Field Gradients. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, (2), 93-158. 10. Sreerama, N.; Woody, R. W., Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Comparison of Contin, Selcon, and Cdsstr Methods with an Expanded Reference Set. Anal. Biochem. 2000, 287, (2), 252-260. 11. Berne, B. J.; Pecora, R., Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics. Courier Corporation: 2000. 12. Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E., Reversible Unfolding of Individual Titin Immunoglobulin Domains by Afm. Science 1997, 276, (5315), 1109-1112. 13. Zhou, Y. L.; Hu, N. F.; Zeng, Y. H.; Rusling, J. F., Heme Protein-Clay Films: Direct Electrochemistry and Electrochemical Catalysis. Langmuir 2002, 18, (1), 211-219. 14. Dziri, L.; Desbat, B.; Leblanc, R. M., Polarization-Modulated Ft-Ir Spectroscopy Studies of Acetylcholinesterase Secondary Structure at the Air-Water Interface. J. Am. Chem. Soc. 1999, 121, (41), 9618-9625. 15. Niemeyer, C. M., Nanoparticles, Proteins, and Nucleic Acids: Biotechnology Meets Materials Science. Angew. Chem. Int. Edit. 2001, 40, (22), 4128-4158. 16. Tamm, L. K.; Tatulian, S. A., Infrared Spectroscopy of Proteins and Peptides in Lipid Bilayers. Q. Rev. Biophys. 1997, 30, (4), 365-429. 17. Barth, A.; Zscherp, C., What Vibrations Tell Us About Proteins. Q. Rev. Biophys. 2002, 35, (4), 369-430. 18. Wang, J.; Chen, X.; Clarke, M. L.; Chen, Z., Detection of Chiral Sum Frequency Generation Vibrational Spectra of Proteins and Peptides at Interfaces in Situ. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, (14), 4978-4983. 19. Rocha-Mendoza, I.; Yankelevich, D. R.; Wang, M.; Reiser, K. M.; Frank, C. W.; Knoesen, A., Sum Frequency Vibrational Spectroscopy: The Molecular Origins of the Optical Second-Order Nonlinearity of Collagen. Biophys. J. 2007, 93, (12), 4433-4444. 20. Nguyen, K. T., An Electronically Enhanced Chiral Sum Frequency Generation Vibrational Spectroscopy Study of Lipid-Bound Cytochrome C. Chem. Commun. 2015, 51, (1), 195-197.

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