Chiral sum frequency generation spectroscopy for characterizing protein secondary structures at interfaces

In situ and real-time characterization of protein secondary structures at interfaces is important in biological and bioengineering sciences, yet remains technically challenging. In this study, we used chiral sum frequency generation (SFG) spectroscopy to establish a set of vibrational optical markers for characterizing protein secondary structures at interfaces. We discovered that the N-H stretches along the peptide backbones of α-helices can be detected in chiral SFG spectra. We further observed that the chiral vibrational signatures of the N-H stretch together with the peptide amide I are unique to α-helix, β-sheet, and random coil at interfaces. Using these chiral vibrational signatures, we studied the aggregation of human islet amyloid polypeptide (hIAPP), which is implicated in type II diabetes. We observed in situ and in real time the misfolding of hIAPP from random coils to α-helices and then β-sheets upon interaction with a lipid-water interface. Our findings show that chiral SFG spectroscopy is a powerful tool to follow changes in protein conformations at interfaces and identify interfacial protein secondary structures that elude conventional techniques.     

Multiple orientation of melittin inside a single lipid bilayer determined by combined vibrational spectroscopic studies

Despite the availability of several mature structure determination techniques for bulk proteins, determination of structural and orientational information of interfacial proteins, e.g., in cell membranes or on biomaterial surfaces, remains a difficult problem. We combine sum frequency generation (SFG) vibrational spectroscopy with attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) to investigate the orientation of alpha-helical peptides reconstituted in substrate supported lipid bilayers. Melittin was chosen as a model for alpha-helical peptides, and its orientation when interacting with a supported 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) bilayer has been examined. Through polarization analysis using amide I signals obtained from both SFG and ATR-FTIR measurements, the orientation distribution of melittin inside a DPPG bilayer was deduced using several trial distribution functions. Melittin was modeled as either an ideal helix or a helix with a bent structure. It was found that a simple distribution function such as a delta-distribution or a Gaussian distribution was not adequate to describe the melittin orientation distribution inside a DPPG bilayer. Instead, two populations of melittin, corresponding to two melittin-bilayer association states, could be used to interpret the experimentally observed result. The method employed in this study demonstrates the feasibility of acquiring a more accurate orientation distribution of peptides/proteins in situ using a combination of vibrational spectroscopic techniques without exogenous labeling.     

Probing alpha-helical and beta-sheet structures of peptides at solid/liquid interfaces with SFG

We demonstrated that sum frequency generation (SFG) vibrational spectroscopy can distinguish different secondary structures of proteins or peptides adsorbed at solid/liquid interfaces. The SFG spectrum for tachyplesin I at the polystyrene (PS)/solution interface has a fingerprint peak corresponding to the B1/B3 mode of the antiparallel beta-sheet. This peak disappeared upon the addition of dithiothreitol, which can disrupt the beta-sheet structure. The SFG spectrum indicative of the MSI594 alpha-helical structure was observed at the PS/MSI594 solution interface. This research validates SFG as a powerful technique for revealing detailed secondary structures of interfacial proteins and peptides.     

Quantifying the ordering of adsorbed proteins in situ

We have investigated the orientation and conformation of protein molecules at the polystyrene (PS)/protein solution interface using sum frequency generation (SFG) vibrational spectroscopy, supplemented by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). In this research, we studied fibrinogen as a model protein. SFG studies indicate that fibrinogen adopts a bent structure after adsorbing to the PS surface. A broad orientation distribution of fibrinogen coiled-coils at the interface has been quantified by combining SFG and ATR-FTIR measurements. Error analysis for such a deduced distribution was carried out. This research demonstrates that quantitative structural information such as orientational and conformational ordering of proteins at interfaces can be studied using SFG supplemented by other spectroscopic techniques.     

Solvent effect and time-dependent behavior of C-terminus-cysteine-modified cecropin P1 chemically immobilized on a polymer surface

Sum frequency generation (SFG) vibrational spectroscopy has been applied to the investigation of peptide immobilization on a polymer surface as a function of time and peptide conformation. Surface immobilization of biological molecules is important in many applications such as biosensors, antimicrobial materials, biobased fuel cells, nanofabrication, and multifunctional materials. Using C-terminus-cysteine-modified cecropin P1 (CP1c) as a model, we investigated the time-dependent immobilization behavior in situ in real time. In addition, potassium phosphate buffer (PB) and mixtures of PB and trifluoroethanol were utilized to examine the effect of peptide secondary structure on CP1c immobilization to polystyrene maleimide (PS-MA). The orientation of immobilized CP1c on PS-MA was determined using polarized SFG spectra. It was found that the peptide solution concentration, solvent composition, and assembly state (monomer vs dimer) prior to immobilization all influence the orientation of CP1c on a PS-MA surface. The detailed relationship between the interfacial peptide orientation and these immobilization conditions is discussed.     

Conformational changes of fibrinogen after adsorption

The adsorption behavior of fibrinogen to two biomedical polyurethanes and a perfluorinated polymer has been investigated. Changes in the secondary structure of adsorbed fibrinogen were monitored using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and sum frequency generation vibrational spectroscopy (SFG). SFG measurements were performed in the amide I range as well as in the C-H/N-H stretching range. Amide I signals from SFG demonstrate that fibrinogen has post-adsorption conformational changes that are dependent upon the polymer surface properties. For example, strong attenuation of the amide I and N-H stretching signals with increasing residence time was observed for fibrinogen adsorbed to poly(ether urethane) but not for the other two polymers. This change is not readily observed by ATR-FTIR. Differences in the observed spectral changes for fibrinogen adsorbed to each polymer are explained by different initial binding mechanisms and post-adsorption conformational changes.     

Deduction of structural information of interfacial proteins by combined vibrational spectroscopic methods

We demonstrate both theoretically and experimentally that the combination of vibrational spectroscopic techniques on samples can be used to deduce more detailed structural information of interfacial proteins and peptides. Such an approach can be used to elucidate structures of proteins or peptides at interfaces, such as at the solid/liquid interface or in cell membranes. We also discuss that the controlled perturbations may provide more measured parameters for structural studies on such proteins and peptides. In this paper, we will demonstrate that optical spectroscopic techniques such as polarized Fourier transform infrared spectroscopy (FTIR), sum frequency generation (SFG) vibrational spectroscopy, and higher order nonlinear vibrational spectroscopies can be used to deduce different and complementary structural information of molecules at interfaces (e.g., orientation information of certain functional groups and secondary structures of interfacial proteins). Also, we believe that controlled perturbations on samples, such as variation of sample temperature, application of electrical fields, and alternation of substrate roughness, can provide more detailed information regarding the interfacial structures of proteins and peptides. The development of nonlinear vibrational spectroscopies, such as SFG and four-wave mixing vibrational spectroscopy, to examine interfacial protein and peptide structures, and introduction of external perturbations on samples should be able to substantially advance our knowledge in understanding structures and thus functions of proteins and peptides at interfaces.     

Ordered adsorption of coagulation factor XII on negatively charged polymer surfaces probed by sum frequency generation vibrational spectroscopy

Electrostatic interactions between negatively charged polymer surfaces and factor XII (FXII), a blood coagulation factor, were investigated by sum frequency generation (SFG) vibrational spectroscopy, supplemented by several analytical techniques including attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), quartz crystal microbalance (QCM), ζ-potential measurement, and chromogenic assay. A series of sulfonated polystyrenes (sPS) with different sulfonation levels were synthesized as model surfaces with different surface charge densities. SFG spectra collected from FXII adsorbed onto PS and sPS surfaces with different surface charge densities showed remarkable differences in spectral features and especially in spectral intensity. Chromogenic assay experiments showed that highly charged sPS surfaces induced FXII autoactivation. ATR-FTIR and QCM results indicated that adsorption amounts on the PS and sPS surfaces were similar even though the surface charge densities were different. No significant conformational change was observed from FXII adsorbed onto surfaces studied. Using theoretical calculations, the possible contribution from the third-order nonlinear optical effect induced by the surface electric field was evaluated, and it was found to be unable to yield the SFG signal enhancement observed. Therefore it was concluded that the adsorbed FXII orientation and ordering were the main reasons for the remarkable SFG amide I signal increase on sPS surfaces. These investigations indicate that negatively charged surfaces facilitate or induce FXII autoactivation on the molecular level by imposing specific orientation and ordering on the adsorbed protein molecules. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The effect of surface coverage on conformation changes of bovine serum albumin molecules at the air-solution interface detected by sum frequency generation vibrational spectroscopy

The air-BSA solution interface has been investigated by various techniques for years. From these studies we know that BSA molecules segregate at the BSA solution-air interface, and the surface coverage increases with the increase of the bulk solution concentration. However, questions still remain as to whether the protein changes conformation, orientation, or a combination of the two upon adsorption. In this paper, by using sum frequency generation (SFG) vibrational spectroscopy we found that the conformation of interfacial BSA molecules changes dramatically at the solution-air interface, compared to that of the native BSA in solution. The hydrophobic methyl groups of BSA molecules at this interface tend to align along the surface normal. The degree of such conformational changes of surface BSA molecules depend on the surface coverage, indicating that the protein-protein interaction plays a very important role in determining the conformation of interfacial protein molecules. At very low surface concentration, the adsorbed BSA molecules unfold substantially. Our results can provide a molecular interpretation of results obtained from other studies such as protein layer thickness and surface tension measurements of protein solution.     

Observing a molecular knife at work

Sum frequency generation (SFG) vibrational spectroscopy has been employed to study the molecular interactions between a single substrate supported lipid bilayer and an amphiphilic antibiotic compound 1, with a design based on the common structural motif of natural antimicrobial peptides. The interfacial sensitivity of SFG allows real-time in situ monitoring of ordering changes in both leaflets of the bilayer and orientation of 1 simultaneously. A critical concentration of about 0.8 microg/mL of 1 is found, above which the inner leaflet of the bilayer is significantly perturbed. This concentration corresponds well to the minimum inhibition concentration of 1 that is obtained from bacterial experiments. Orientation of 1 in the bilayer is shown to be perpendicular to the bilayer surface, in agreement with simulation results. SFG can be developed into a very informative technique for studying the cell membrane and the interactions of membrane-active molecules.     

Detection of amide I signals of interfacial proteins in situ using SFG

In this Communication, we demonstrate the novel observation that it is feasible to collect amide signals from polymer/protein solution interfaces in situ using sum frequency generation (SFG) vibrational spectroscopy. Such SFG amide signals allow for acquisition of more detailed molecular level information of entire interfacial protein structures. Proteins investigated include bovine serum albumin, mussel protein mefp-2, factor XIIa, and ubiquitin. Our studies indicate that different proteins generate different SFG amide signals at the polystyrene/protein solution interface, showing that they have different interfacial coverage, secondary structure, or orientation.     

Observing a model ion channel gating action in model cell membranes in real time in situ: membrane potential change induced alamethicin orientation change

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/3(10)-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 3(10)-helix at the C-terminus tilt at about 72° (θ(1)) and 50° (θ(2)) versus the surface normal, respectively. When pH increases to 11.9, θ(1) and θ(2) 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 θ(1) 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.     

Molecular interactions of proteins and peptides at interfaces studied by sum frequency generation vibrational spectroscopy

Interfacial peptides and proteins are critical in many biological processes and thus are of interest to various research fields. To study these processes, surface sensitive techniques are required to completely describe different interfacial interactions intrinsic to many complicated processes. Sum frequency generation (SFG) spectroscopy has been developed into a powerful tool to investigate these interactions and mechanisms of a variety of interfacial peptides and proteins. It has been shown that SFG has intrinsic surface sensitivity and the ability to acquire conformation, orientation, and ordering information about these systems. This paper reviews recent studies on peptide/protein-substrate interactions, peptide/protein-membrane interactions, and protein complexes at interfaces and demonstrates the ability of SFG on unveiling the molecular pictures of complicated interfacial biological processes.     

空气/水界面水分子的取向和运动

Here we report a quantitative study of the orientational structure and motion of water molecule at the air/water interface. Analysis of Sum Frequency Generation (SFG) vibrational peak of the free O-H stretching band at 3700 cm-1 in four experimental configurations showed that orientational motion of water molecule at air/water interface is libratory within a limited angular range. The free OH bond of the interfacial water molecule is tilted around 33° from the interface normal and the orientational distribution or motion width is less than 15°. This picture is significantly different from the previous conclusion that the interfacial water molecule orientation varies over a broad range within the ultrafast vibrational relaxation time, the only direct experimental study concluded for ultrafast and broad orientational motion of a liquid interface by Wei et al.(Phys. Rev. Lett. 86, 4799, (2001)) using single SFG experimental configuration.     

Recent progress in theoretical analysis of vibrational sum frequency generation spectroscopy

This article summarizes the computational analysis of the vibrational sum frequency generation (SFG) spectroscopy with molecular dynamics simulation. The analysis allows direct comparison of experimental SFG spectra and microscopic interface structure obtained by molecular simulation, and thereby obviates empirical fitting procedures of the observed spectra. In the theoretical formulation, the frequency-dependent nonlinear susceptibility of an interface is calculated in two ways, based on the energy representation and time-dependent representation. The application to aqueous interfaces revealed a number of new insights into the local structure of electrolyte interfaces and the interpretation of SFG spectroscopy.     

Vibrational spectral signatures of peptide secondary structures: N-methylation and side chain hydrogen bond in cyclosporin A

Molecular dynamics simulations are performed to explore important conformations of cyclosporin A, an immunosuppressive cyclic undecapeptide drug, in different media including gas-phase, chloroform, and acetonitrile. Density functional theory calculations are used to refine the low-lying conformers and to predict their infrared and vibrational circular dichroism spectra. Vibrational spectral signatures in the important amide II, I, and A regions are identified for typical peptide secondary structures including β-turn (type II' or I), antiparallel β-sheet (flat or twisted), inverse γ-turn, N-methylated peptide bond, and side chain H-bond. New insights into the spectral signatures of secondary structures especially with N-methylation and side chain hydrogen bond are provided, which can be very useful for further peptide conformation analysis in general.     

Orientational motions of vibrational chromophores in molecules at the air/water interface with time-resolved sum frequency generation

The first time-resolved experiments in which interfacial molecules are pumped to excited electronic states and probed by vibrational sum frequency generation (SFG) are reported. This method was used to measure the out-of-plane rotation dynamics, i.e. time dependent changes in the polar angle, of a vibrational chromophore of an interfacial molecule. The chromophore is the carbonyl group, the rotation observed is that of the -C=O bond axis, with respect to the interfacial normal, and the interfacial molecule is coumarin 314 (C314) at the air/water interface. The orientational relaxation time was found to be 220+/-20 ps, which is much faster than the orientational relaxation time of the permanent dipole moment axis of C314 at the same interface, as obtained from pump-second harmonic probe experiments. Possible effects on the rotation of the -C=O bond axis due to the carbonyl group hydrogen bonding with interfacial water are discussed. From the measured equilibrium orientation of the permanent dipole moment axis and the carbonyl axis, and knowledge of their relative orientation in the molecule, the absolute orientation of C314 at the air/water interface is obtained.     

Effect of dehydration on the interfacial water structure at a charged polymer surface: negligible χ(3) contribution to sum frequency generation signal

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 χ(ppp)/χ(ssp) 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.     

Surface plasma treatment effects on the molecular structure at polyimide/air and buried polyimide/epoxy interfaces

Polyimides are widely used as chip passivation layers and organic substrates in microelectronic packaging. Plasma treatment has been used to enhance the interfacial properties of polyimides, but its molecularmechanism is not clear. In this research, the effects of polyimide surface plasma treatment on the molecular structures at corresponding polyimide/air and buried polyimide/epoxy interfaces were investigated in situ using sum frequency generation (SFG) vibrational spectroscopy. SFG results show that the polyimide backbone molecular structure was different at polyimide/air and polyimide/epoxy interfaces before and after plasma treatment. The different molecular structures at each interface indicate that structural reordering of the polyimide backbone occurred as a result of plasma treatment and contact with the epoxy adhesive. Furthermore, quantitative orientation analysis indicated that plasma treatment of polyimide surfaces altered the twist angle of the polyimide backbone at corresponding buried polyimide/epoxy interfaces. These SFG results indicate that plasma treatment of polymer surfaces can alter the molecular structure at corresponding polymer/air and buried polymer interfaces.     

Real-Time Observation of Protein Transport across Membranes by Femtosecond Sum Frequency Generation Vibrational Spectroscopy?

Characterization of conformation kinetics of proteins at the interfaces is crucial for understanding the biomolecular functions and the mechanisms of interfacial biological action. But it requires to capture the dynamic structures of proteins at the interfaces with sufficient structural and temporal resolutions. Here, we demonstrate that a femtosecond sum frequency generation vibrational spectroscopy (SFG-VS) system developed by our group provides a powerful tool for monitoring the real-time peptide transport across the membranes with time resolution of less than one second. By probing the real-time SFG signals in the amide Ⅰ and amide A bands as WALP23 interacts with DMPG lipid bilayer, it is found that WALP23 is initially absorbed at the gel-phase DMPG bilayer with a random coil structure. The absorption of WALP23 on the surface leads to the surface charge reversal and thus changes the orientation of membrane-bound water. As the DMPG bilayer changes from gel phase into fluid phase, WALP23 inserts into the fluid-phase bilayer with its N-terminal end moving across the membrane, which causes the membrane dehydration and the transition of WALP23 conformation from random coil to mixed helix/loop structure and then to pure α-helical structure. The established system is ready to be employed in characterizing other interfacial fast processes, which will be certainly helpful for providing a clear physical picture of the interfacial phenomena.