The molecular structure and dynamics of organic molecules at the aqueous interface have attracted a number of investigations owing to their importance and specific nature. However, there are relatively few studies on the direct characterization of the molecular interactions at the air/water interface because they are extremely difficult to measure in experiments. In this study, we use dibutyl ester molecules (R1CO2R2O2CR1) as a model of organic molecules, and investigate their molecular structure and interactions using sum frequency generation vibrational spectroscopy. We demonstrate that the molecular interactions can be estimated by measuring the intensity ratio of the symmetric stretching (ν1) and Fermi resonant bands (2ν2) of methyl groups. Here, dibutyl ester molecules are widely used as plasticizers in polymers to improve the properties of the plastics and polymers. It is found that the orientation angles of the tailed methyl groups at the air/water interface decrease from 34° to 19° when the chain length of R2 increases from 0 to 8. The total intermolecular interactions of the dibutyl ester molecules decrease as the chain length of R2 increases because the van der Waals interactions between the hydrocarbon chains increase, while the hydrogen bond interactions between the carbonyl group and water molecules decrease. Our study demonstrates the stability of ester-based plasticizers in polymers can be well predicted from the intensity ratio of the ν1 and 2ν2 bands of methyl group. Such an intensity ratio can be thus used as an effective vibrational optical ruler for characterizing molecular interactions between plasticizers and polymers.

AbstractVibrational energy transfer (VET) of proteins at cell membrane plays critical roles in controlling the protein functionalities, but its detection is very challenging. By using a surface-sensitive femtosecond time-resolved sum-frequency generation vibrational spectroscopy with infrared pump, the detection of the ultrafast VET in proteins at cell membrane has finally become possible. The vibrational relaxation time of the N−H groups is determined to be 1.70(±0.05) ps for the α-helix located in the hydrophobic core of the lipid bilayer and 0.9(±0.05) ps for the membrane-bound β-sheet structure. The N−H groups with strong hydrogen bonding gain faster relaxation time. By pumping the amide A band and probing amide I band, the vibrational relaxation from N−H mode to C=O mode through two pathways (direct coupling and through intermediate states) is revealed. The ratio of the pathways depends on the NH⋅⋅⋅O=C hydrogen-bonding strength. Strong hydrogen bonding favors the coupling through intermediate states.

AbstractVibrational energy transfer (VET) of proteins at cell membrane plays critical roles in controlling the protein functionalities, but its detection is very challenging. By using a surface-sensitive femtosecond time-resolved sum-frequency generation vibrational spectroscopy with infrared pump, the detection of the ultrafast VET in proteins at cell membrane has finally become possible. The vibrational relaxation time of the N−H groups is determined to be 1.70(±0.05) ps for the α-helix located in the hydrophobic core of the lipid bilayer and 0.9(±0.05) ps for the membrane-bound β-sheet structure. The N−H groups with strong hydrogen bonding gain faster relaxation time. By pumping the amide A band and probing amide I band, the vibrational relaxation from N−H mode to C=O mode through two pathways (direct coupling and through intermediate states) is revealed. The ratio of the pathways depends on the NH⋅⋅⋅O=C hydrogen-bonding strength. Strong hydrogen bonding favors the coupling through intermediate states.

A good understanding of membrane protein folding at the molecular level requires an effective means to determine the dynamical structural changes on coil-to-helix transition within the cell membrane and as yet remains challenging. Herein, we demonstrate that the amide III spectral signals of the protein backbone, generated in the sum frequency generation vibrational spectroscopy, are a powerful tool to probe the protein folding processes within the membrane in situ, in real time, and without exogenous labels. The amide III signals are capable of separating the spectral profiles of the random-coil and α-helical structures at the interface. The intensity ratio of coil and helix peaks becomes a prime indicator that allows one to directly capture the dynamical change of the coil–helix transition. With this approach, using pardaxin as a model, the influence of lipid charge on the peptide folding degree at the cell membrane surface has been nicely elucidated. It is evident that the negative charge of the lipid increases the folding degree of pardaxin upon interfacial adsorption and promotes the formation of α-helical structure during the insertion of peptides into the lipid bilayer. This robust spectral approach can thus greatly enhance our ability to monitor the dynamics of membrane proteins in a real cell environment in situ.

Understanding the transport behavior of the cholesterol molecules within a cell membrane is a key challenge in cell biology at present. Here, we have applied sum frequency generation vibrational spectroscopy to characterize the transport and organization of cholesterol in different kinds of planar solid-supported lipid bilayers by combining achiral- and chiral-sensitive polarization measurements. This method allows us to distinguish the organization of cholesterol in tail-to-tail, head-to-tail, head-to-head, and side-by-side manners. It is found that the movement of cholesterol in the lipid bilayer largely depends on the flip-flop rate of the phospholipid. The flip-flop dynamics of the phospholipid and cholesterol are synchronous. In the solid-supported zwitterionic phosphocholine lipid bilayer, the cholesterol molecules flip quickly from the distal leaflet to the neutral proximal leaflet of the bilayer and form tail-to-tail organization on both leaflets. The phosphocholine lipid and cholesterol show the same flip-flop rate. However, when the proximal leaflet is prepared using negative glycerol phospholipids, cholesterol organizes itself by mainly forming an α–β structure on the distal leaflet. Because of the strong interaction between the glycerol phospholipid and the substrate, no or only partial cholesterol molecules flip from the distal leaflet to the negatively charged proximal leaflet. However, the cholesterol molecules undergo flip-flop in the presence of salt solution because the ions weaken the interaction between the negative phospholipid and the substrate.

Membrane domain formation plays a key role in various cellular functions and biological events. Lateral accumulation of lipids and proteins in biological membranes is one of the most important factors that control the domain formation. However, compared to numerous reports on the lipid aggregation or accumulation formed in the membranes composed of multiple components of lipids and cholesterol, the lipid accumulation in one-component phospholipid bilayer system is still rare. In this study, we demonstrate that short peptides can induce the lipid accumulation in a single-component zwitterionic lipid bilayer. By investigating the interaction between a short peptide of mastoparan (MP, a G-protein-activating peptide) and neutral phosphocholine lipid bilayers using sum frequency generation vibrational spectroscopy (SFG-VS), we have found that MP can cause a local accumulation of lipid molecules at the outer leaflet of the lipid bilayer, resulting in more than 10 times intensity increase in the signals from the CD3 vibrational modes with respect to that of lipid monolayer at the air surface. We have validated that the lipid accumulation behavior originates from a specific hydrophobic-mismatching interaction in which the peptide is too short to span the lipid bilayer. Our results suggest that other mechanisms that do not involve perforation exist for the interactions between peptides and membranes. This finding broadens the range of systems and our basic understanding on lipid accumulation.

Dehydration of a surface is the first step for the interaction between biomolecules and the surface. In this study, we systematically investigated the influence of cholesterol analog 6-ketocholestanol (6-KC) on the dehydration of model cell membrane, using sum frequency generation vibrational spectroscopy. In pure DI water environment, two separate dehydration dynamic components were observed in neutrally charged and isotopically labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and positively charged 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine(chloride salt) (DMEPC) bilayer: a large-amplitude fast component and a small-amplitude slow component, which originated from the water molecules with a weak and a strong water-membrane bound strengths, respectively. Dehydration of a negatively charged mixed DMPC/DMPG bilayer lead to the membrane-bound water being reorganized to ordered structures quickly. It is evident that the water-membrane bound strengths depend largely on the charge status of the lipid and has an order of neutrally charged membrane≪positively charged membrane≪negatively charged membrane. In an ionic environment, KCl solution can not only dehydrate DMPC bilayer, but also prevent the 6-KC from further dehydrating this model cell membrane. We observed that the dehydration dynamics behavior of DMPC bilayer in the presence of the chaotropic anions is similar to that of the negatively charged DMPG bilayer because of the penetration of chaotropic anions into the DMPC bilayer. The degree of dehydration difficulty in kosmotropic anions follows a Hofmeister series and linearly correlates with the hydration Gibbs free energy of the anions. Our results provide a molecular basis for the interpretation of the Hofmeister effect of kosmotropic anions on ion transport proteins.

Accurate determination of intermolecular interaction forces at the surface and the interface is essential to identify the nature of interfacial phenomena such as absorption, interfacial assembly, and specific ion effect, but it still represents a major technical challenge. In this study, we proposed a novel method to deduce the interfacial interaction forces by using surface-sensitive second-order Fermi resonant signals, generated in sum frequency generation vibrational spectroscopy (SFG-VS). By investigating the influence of lipid chain length and intermolecular distance on the Fermi resonant signals of phospholipid monolayer at the air/CaF2 surface and the air/water interface, a linear correlation between the Fermi resonant intensity ratio and the dominated interactions in the lipid monolayer has been observed. It implies that the amplitude of the intensity ratio can be used as an effective in situ vibrational optical ruler to characterize the total intermolecular interaction forces at the surface and the interface. Such a relationship further enables us to elucidate the specific ion effects on the interfacial interactions, allowing us to identify different contributions from van der Waals, electrostatic, and hydration interactions. This study clearly demonstrates the power of the second-order Fermi resonant signals for evaluating the interfacial interaction forces in situ and in real time.

Molecular insight into the interactions of two-dimensional (2D) materials at the interface is essential to understand the functionality of interfacial molecular devices. Yet it still remains elusive so far. Fermi resonant interaction is highly sensitive to the total molecular interactions. In this study, we used lipid 1,2-dimyristoyl-sn-glycero-3 -phospho-(1′-rac-glycerol) (sodium salt) (DMPG) monolayer as a model, and performed a systematic study to investigate the Fermi resonant interactions of 2D materials at the interface during liquid-expanded (LE) to liquid-condensed (LC) phase transition using multiplexed-polarization sum frequency generation vibrational spectroscopy (SFG-VS). It is found that the ratio (R1) between Fermi resonance and symmetric stretching mode of the tailed methyl groups sharply decreases during the phase transition. The sharp drop of R1 originates from the nonsynchronous change of the tail and head groups of the lipid. The tailed CH3 groups of DMPG locally accumulate at the air/water interface during LE–LC phase transition while the head glycerol groups do not. The local aggregation of the methyl groups strengthens the van der Waals (vdW) interaction, leading to the decrease of the total intermolecular interactions and the drop of the ratio of R1. However, such phenomena are not observed at the air/KCl solution (0.3M) interface.

Accurate determination of protein structures at the interface is essential to understand the nature of interfacial protein interactions, but it can only be done with a few, very limited experimental methods. Here, we demonstrate for the first time that sum frequency generation vibrational spectroscopy can unambiguously differentiate the interfacial protein secondary structures by combining surface-sensitive amide I and amide III spectral signals. This combination offers a powerful tool to directly distinguish random-coil (disordered) and α-helical structures in proteins. From a systematic study on the interactions between several antimicrobial peptides (including LKα14, mastoparan X, cecropin P1, melittin, and pardaxin) and lipid bilayers, it is found that the spectral profiles of the random-coil and α-helical structures are well separated in the amide III spectra, appearing below and above 1260 cm–1, respectively. For the peptides with a straight backbone chain, the strength ratio for the peaks of the random-coil and α-helical structures shows a distinct linear relationship with the fraction of the disordered structure deduced from independent NMR experiments reported in the literature. It is revealed that increasing the fraction of negatively charged lipids can induce a conformational change of pardaxin from random-coil to α-helical structures. This experimental protocol can be employed for determining the interfacial protein secondary structures and dynamics in situ and in real time without extraneous labels.

Sum frequency generation vibrational spectroscopy (SFG-VS) has been demonstrated to be a powerful technique to study the interfacial structures and interactions of biomolecules at the molecular level. Yet most previous studies mainly collected the SFG spectra in the frequency range of 1500–4000 cm−1, which is not always sufficient to describe the detailed interactions at surface and interface. Thorough knowledge of the complex biophysicochemical interactions between biomolecules and surface requires new ideas and advanced experimental methods for collecting SFG vibrational spectra. We introduced some advanced methods recently exploited by our group and others, including (1) detection of vibration modes in the fingerprint region; (2) combination of chiral and achiral polarization measurements; (3) SFG coupled with surface plasmon polaritons (SPPs); (4) imaging and microscopy approaches; and (5) ultrafast time-resolved SFG measurements. The technique that we integrated with these advanced methods may help to give a detailed and high-spatial-resolution 3D picture of interfacial biomolecules.

Cholesterol organization and transport within a cell membrane are essential for human health and many cellular functions yet remain elusive so far. Using cholesterol analogue 6-ketocholestanol (6-KC) as a model, we have successfully exploited sum frequency generation vibrational spectroscopy (SFG-VS) to track the organization and transport of cholesterol in a membrane by combining achiral-sensitive ssp (ppp) and chiral-sensitive psp polarization measurements. It is found that 6-KC molecules are aligned at the outer leaflet of the DMPC lipid bilayer with a tilt angle of about 10°. 6-KC organizes itself by forming an α–β structure at low 6-KC concentration and most likely a β–β structure at high 6-KC concentration. Among all proposed models, our results favor the so-called umbrella model with formation of a 6-KC cluster. Moreover, we have found that the long anticipated flip-flop motion of 6-KC in the membrane takes time to occur, at least much longer than previously thought. All of these interesting findings indicate that it is critical to explore in situ, real-time, and label-free methodologies to obtain a precise molecular description of cholesterol’s behavior in membranes. This study represents the first application of SFG to reveal the cholesterol–lipid interaction mechanism at the molecular level.Keywords: cellular organization and transport; cholesterol; flip-flop; label-free; molecular level; sum frequency generation;

Phosphate ion is one of the most important anions present in the intracellular and extracellular fluid. It can form strongly hydrogen-bonded and salt-bridged complexes with arginine and lysine to activate the voltage gated channel protein. A molecular-level insight into how the phosphate anions mediate the interaction between peptides and cell membrane is critical to understand membrane-bound peptide actions. In this study, sum frequency generation vibrational spectroscopy (SFG-VS) has been applied to characterize interactions between mastoparan (MP, a G-protein-activating peptide) and different charged lipid bilayers in situ. It is found that phosphate ions can greatly promote the association of MP with lipid bilayers and accelerate the conformation transition of membrane-bound MP from aggregation into α-helical structure. In phosphate buffer solution, MP can insert not only into negatively and neutrally charged lipid bilayers but also into positively charged lipid bilayers. In neutrally and negatively charged lipid bilayers, the tilt angle of α-helical structure becomes smaller with increasing buffer concentration, while MP adopts a multiple orientation distribution in the positively charged lipid bilayer. MP interacts with lipid bilayers in the salt solution environment most likely by formation of toroidal pores inside the bilayer matrix. Results from our studies will provide insight into the MP action mechanism and offer some ideas to deliver exogenous protein into the cytosol.

Insertion of short peptides into the cell membrane is energetically unfavorable and challenges the commonly accepted hydrophobic matching principle. Yet there has been evidence that many short peptides can penetrate into the cells to perform the biological functions in salt solution. On the basis of the previous study ( J. Phys. Chem. C 2013, 117, 11095−11103), here we further performed a systematic study on the interaction of mastoparan with various neutral lipid bilayers with different lipid chain lengths in situ to examine the hydrophobic matching principle in different aqueous salt environments using sum frequency generation vibrational spectroscopy. It is found that the hydrophobic matching is the dominant driving force for the association of MP with a lipid bilayer in a pure water environment. However, in a kosmotropic ion environment, the hydration of ions can overcome the hydrophobic mismatching effects, leading to the insertion of MP into lipid bilayers with much longer hydrophobic lengths. When the hydrophobic thickness of the bilayer is much longer than MP’s hydrophobic length, MP diffuses on a single monolayer, rather than spanning the bilayer to prevent the exposure of the hydrophilic part of MP to the lipid hydrophobic moiety. Findings from the present study suggest that the interaction between the positively charged choline group of a lipid and kosmotropic ions could be an important step for effective peptide insertion into a cell membrane. Results from our studies will provide an insight into how the short peptides form the ion channel in a thick membrane and offer some ideas for cellular delivery.

Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split α/310-helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer and was measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH = 6.7 the α-helix at the N-terminus and the 310-helix at the C-terminus tilt at about 72° (θ1) and 50° (θ2) versus the surface normal, respectively. When pH increases to 11.9, θ1 and θ2 decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ1 for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and the bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes.

The complex structure of water and its interactions with solid surfaces require the development of multiple vibrational spectroscopic measurements to study the molecular structure of interfacial water and bulk water near the solid surface. In this study, a newly developed compatible multiple nonlinear vibrational spectroscopy system has been applied to investigate the molecular structure of water in the interfacial region of an ionic solid (CaF2 substrate) and bulk isotopic D2O–HOD–H2O mixtures. Using this compatible system, the sum frequency generation (SFG) vibrational spectra and infrared-infrared-visible three-pump-field four-wave-mixing (IIV-TPF-FWM) spectra of the same water molecules can be collected at the same experimental geometry. It is found that SFG and IIV-TPF-FWM can be used to characterize the molecular structures of interfacial water and bulk water molecules at an interfacial distance below 42 nm, respectively. SFG and IIV-TPF-FWM results both suggest an intramolecular vibrational coupling dominates the spectra. The results achieved by this method are helpful to clarify the origination of the vibrational coupling of the interfacial water as well as the bulk water near the solid surface.

Protein aggregation is associated with many “protein deposition diseases”. A precise molecular detail of the conformational transitions of such a membrane-associated protein structure is critical to understand the disease mechanism and develop effective treatments. One potential model peptide for studying the mechanism of protein deposition diseases is prion protein fragment [118–135] (PrP118–135), which shares homology with the C-terminal domain of the Alzheimer’s β-amyloid peptide. In this study, sum frequency generation vibrational spectroscopy (SFG-VS) has been applied to characterize interactions between PrP118–135 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) lipid bilayer in situ. The conformation change and orientation of PrP118–135 in lipid bilayers have been determined using SFG spectra with different polarization combinations. It is found that low-concentration PrP118–135 predominantly adopts α-helical structure but with tiny β-sheet structure. With the PrP118–135 concentration increasing, the molecular number ratio of parallel β-sheet structure increases and reaches about 44% at a concentration of 0.10 mg/mL, indicating the formation of abnormally folded scrapie isoforms. The α-helical structure inserts into the lipid bilayer with a tilt angle of ∼32° versus the surface normal, while the β-sheet structure lies down on the lipid bilayer with the tilt and twist angle both of 90°. The 3300 cm–1 N–H stretching signal in psp spectra arises from α-helical structure at low PrP concentration and from the β-sheet structure at high PrP concentration. Results from this study will provide an in-depth insight into the early events in the aggregation of PrP in cell membrane.

Interfacial water structure at charged surfaces plays a key role in many physical, chemical, biological, environmental, and industrial processes. Understanding the release of interfacial water from the charged solid surfaces during dehydration process may provide insights into the mechanism of protein folding and the nature of weak molecular interactions. In this work, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by quartz crystal microbalance (QCM) measurements, has been applied to study the interfacial water structure at polyelectrolyte covered surfaces. Poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) chains are grafted on solid surfaces to investigate the change of interfacial water structure with varying surface charge density induced by tuning the solution pH. At pH ≤ 7.1, SFG-VS intensity is linear to the loss of mass of interfacial water caused by the dehydration of PDMAEMA chains, and no reorientation of the strongly bonded water molecules is observed in the light of χppp/χssp ratio. χ(3) contribution to SFG signal is deduced based on the combination of SFG and QCM results. It is the first direct experimental evidence to reveal that the χ(3) has a negligible contribution to SFG signal of the interfacial water at a charged polymer surface.

In-situ and real-time characterization of molecular structure of pH stimuli-responsive assembling systems at interfaces is critical to understand the nature of interfacial driving force and weak molecular interaction behind such reactions and provide important clues to control them in a desired manner. In this study, sum frequency generation vibrational spectroscopy (SFG-VS) has been applied, supplemented by surface pressure (π)–area (A) isotherm measurements, and Brewster angle microscopy images, to investigate the interfacial tautomerism and isomerization reactions occurring in 5-octadecyloxy-2-(2-pyridylazo)phenol (PARC18) monolayer at air/buffer solution interface in situ. The isomerization mechanism was examined by measuring interfacial structure of PARC18 molecule at various subphase pH. Time-dependent change of the SFG intensity of the characteristic band was kinetically measured after spreading PARC18 chloroform solution onto different subphase pH buffer solutions. It was found that hydrazone form prevails on the air/water interface in acidic and neutral conditions while azo form dominates at subphase pH ≥ 11.6. The hydrazone form adopts a planar geometry at pH = 4.5 and 7.0, whereas the azo form adopts a nonplanar cis or cis-like conformation. It was indicated that the trans–cis isomerization processes follow a rotation mechanism. The deprotonation rate constant was deduced to be 0.20–0.42 M–1 s–1 at pH = 10.3–12.6. This is the first reported application of SFG-VS to elucidate the isomerization mechanism and deduce the deprotonation rate constant of azoaromatic compounds at interface. Resulting from this study will aid in a better understanding of the interfacial pH-controlled assembly processes.

In this paper, we designed a compatible multiple nonlinear vibrational spectroscopy system that can be used for recording infrared–visible sum frequency generation vibrational spectra (SFG) and infrared–infrared–visible three-pump-field four-wave-mixing (IIV-TPF-FWM) spectra using a commercial EKSPLA SFG system. This is the first time IIV-TPF-FWM signals were obtained using picosecond laser pulses. We have applied this compatible system to study the surface and vibrational structures of riboflavin molecules (also known as vitamin B2). The SFG spectra of eight polarization combinations have non-vanishing signals. The signals with incoming s-polarized IR are relatively weaker than the signals with incoming p-polarized IR. Under the double resonant conditions, the SFG signals of the conjugated tricyclic ring are greatly enhanced. For the IIV-TPF-FWM spectra with incoming p-polarized IR, only the sspp and pppp polarization combinations have non-vanishing signals. The IIV-TPF-FWM spectra show a very strong peak at 1585 cm−1 that is mainly dominated by the N5–C4a stretch. The method developed in this study will be helpful for researchers, either using a home-built or commercial (EKSPLA) SFG system, to obtain independent and complementary measurements for SFG spectroscopy and more detailed structural information of interfacial molecules.

Protein hydration plays a critical role in protein dynamics and biological processes. Pump−probe transmission measurement has been applied to investigate the hydration effects on the energy relaxation of a heme protein ferric Cytochrome c (Cyt c) film after soret-band photoexcitation. Transient dynamics study indicates that the energy internal conversion time of ∼300 fs is independent of hydration. The vibrationally excited electronic ground-state recovery rates show two transitions at the hydration level of h = 12.4−16.5% and 21.7−23.5%. The first transition occurs at the hydration level for the onset of an increasing ferric Cyt c flexibility while the second transition occurs at the saturated hydration level. The hydration dependence of steady-state electronic absorption spectrum results shows that the Q-band peak is nearly constant in center wavelength, but the line width surprisingly narrows with increasing hydration. For the ∼695 nm absorbance associated with the MET80-Fe bond, the intensity increases with increasing hydration and slightly blue shifts. The 695 nm peak grows rapidly at h = 12.4% and then plateaus at h = 21.7%. This research shows that ∼695 nm absorbance and ground-state recovery rates are sensitive to the hydration of the protein. This study will aid in understanding how hydration modulates the activity of the protein dynamics at a local level.

The molecular structure and dynamics of organic molecules at the aqueous interface have attracted a number of investigations owing to their importance and specific nature. However, there are relatively few studies on the direct characterization of the molecular interactions at the air/water interface because they are extremely difficult to measure in experiments. In this study, we use dibutyl ester molecules (R1CO2R2O2CR1) as a model of organic molecules, and investigate their molecular structure and interactions using sum frequency generation vibrational spectroscopy. We demonstrate that the molecular interactions can be estimated by measuring the intensity ratio of the symmetric stretching (ν1) and Fermi resonant bands (2ν2) of methyl groups. Here, dibutyl ester molecules are widely used as plasticizers in polymers to improve the properties of the plastics and polymers. It is found that the orientation angles of the tailed methyl groups at the air/water interface decrease from 34° to 19° when the chain length of R2 increases from 0 to 8. The total intermolecular interactions of the dibutyl ester molecules decrease as the chain length of R2 increases because the van der Waals interactions between the hydrocarbon chains increase, while the hydrogen bond interactions between the carbonyl group and water molecules decrease. Our study demonstrates the stability of ester-based plasticizers in polymers can be well predicted from the intensity ratio of the ν1 and 2ν2 bands of methyl group. Such an intensity ratio can be thus used as an effective vibrational optical ruler for characterizing molecular interactions between plasticizers and polymers.