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.     

Interfacial orientation and secondary structure change in tachyplesin I: molecular dynamics and sum frequency generation spectroscopy studies

Recent advances in the collection and interpretation of surface-sensitive vibrational spectroscopic measurements have made it possible to study the orientation of peptides and proteins in situ in a biologically relevant environment. However, interpretation of sum frequency generation (SFG) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) vibrational spectroscopy is hindered by the fact that orientation cannot be inferred without some prior knowledge of the protein structure. In this work, molecular dynamics simulations were used to study the interfacial orientation and structural deformation of the short β-sheet peptide tachyplesin I at the polystyrene/water interface. By combining these results with ATR-FTIR and SFG measurements, reasonable agreement was found with the simulation results, suggesting that tachyplesin I lies parallel to the surface, although the simulation results imply a broader distribution of peptide twist angles than could be characterized using available experimental measurements. The interfacial structure was found to be deformable even when disulfide bonds were preserved, and these local deviations from a purely extended β-sheet conformation may be of importance to future developments in the interpretation of SFG and ATR-FTIR spectra.     

Membrane orientation of MSI-78 measured by sum frequency generation vibrational spectroscopy

Antimicrobial peptides (AMPs) selectively disrupt bacterial cell membranes to kill bacteria whereas they either do not or weakly interact with mammalian cells. The orientations of AMPs in lipid bilayers mimicking bacterial and mammalian cell membranes are related to their antimicrobial activity and selectivity. To understand the role of AMP-lipid interactions in the functional properties of AMPs better, we determined the membrane orientation of an AMP (MSI-78 or pexiganan) in various model membranes using sum frequency generation (SFG) vibrational spectroscopy. A solid-supported single 1,2-dipalmitoyl-an-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) bilayer or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) bilayer was used as a model bacterial cell membrane. A supported 1,2-dipalmitoyl-an-glycero-3-phosphocholine (DPPC) bilayer or a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was used as a model mammalian cell membrane. Our SFG results indicate that the helical MSI-78 molecules are associated with the bilayer surface with ～70° deviation from the bilayer normal in the negatively charged gel-phase DPPG bilayer at 400 nM peptide concentration. However, when the concentration was increased to 600 nM, MSI-78 molecules changed their orientation to make a 25° tilt from the lipid bilayer normal whereas multiple orientations were observed for an even higher peptide concentration in agreement with toroidal-type pore formation as reported in a previous solid-state NMR study. In contrary, no interaction between MSI-78 and a zwitterionic DPPC bilayer was observed even at a much higher peptide concentration (～12,000 nM). These results demonstrate that SFG can provide insights into the antibacterial activity and selectivity of MSI-78. Interestingly, the peptide exhibits a concentration-dependent membrane orientation in the lamellar-phase POPG bilayer and was also found to induce toroidal-type pore formation. The deduced lipid flip-flop from SFG signals observed from lipids also supports MSI-78-induced toroidal-type pore formation.     

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.     

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.     

Monitoring orientation and dynamics of membrane-bound melittin utilizing dansyl fluorescence

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. In spite of a number of studies, there is no consensus regarding the orientation of melittin in membranes. In this study, we used a melittin analogue that is covalently labeled at its amino terminal (Gly-1) with the environment-sensitive 1-dimethylamino-5-sulfonylnaphthalene (dansyl) group to obtain information regarding the orientation and dynamics of the amino terminal region of membrane-bound melittin. Our results show that the dansyl group in Dns-melittin exhibits red edge excitation shift in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine, implying its localization in a motionally restricted region of the membrane. This is further supported by wavelength-dependent anisotropy and lifetime changes and time-resolved emission spectra characterized by dynamic Stokes shift, which indicates relatively slow solvent relaxation in the excited state. Membrane penetration depth analysis using the parallax method shows that the dansyl group is localized at a depth of approximately 18 A from the center of the bilayer in membrane-bound Dns-melittin. Further analysis of dansyl and tryptophan depths in Dns-melittin shows that the tilt angle between the helix axis of membrane-bound melittin and the bilayer normal is approximately 70 degrees. Our results therefore suggest that melittin adopts a pseudoparallel orientation in DOPC membranes at low concentration.     

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.     

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.     

Single Lipid Bilayers Constructed on Polymer Cushion Studied by Sum Frequency Generation Vibrational Spectroscopy

Planar solid supported single lipid bilayers on mica, glass, or other inorganic surfaces have been widely used as models for cell membranes. To more closely mimic the cell membrane environment, soft hydrophilic polymer cushions were introduced between the hard inorganic substrate and the lipid bilayer to completely avoid the possible substrate-lipid interactions. In this article, sum frequency generation (SFG) vibrational spectroscopy was used to examine and compare single lipid bilayers assembled on the CaF(2) prism surface and on poly (L-lactic acid) (PLLA) cushion. By using asymmetric lipid bilayers composed of a hydrogenated 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol (DPPG) leaflet and a deuterated 1,2-dipalmitoyl-(d62)-sn-glycerol-3-phosphoglycerol (d-DPPG) leaflet, it was shown that the DPPG lipid bilayers deposited on the CaF(2) and PLLA surfaces have similar structures. SFG has also been applied to investigate molecular interactions between an antimicrobial peptide Cecropin P(1) (CP1) and the lipid bilayers on the above two different surfaces. Similar results were again obtained. This research demonstrated that the hydrophilic PLLA cushion can serve as an excellent substrate to support single lipid bilayers. We believe that it can be an important cell membrane model for future studies on transmembrane proteins, for which the possible inorganic substrate-bilayer interactions may affect the protein structure or function.     

Mapping the orientation of helices in micelle-bound peptides by paramagnetic relaxation waves

Many antimicrobial peptides form alpha-helices when bound to a membrane. In addition, around 80% of residues in membrane-bound proteins are found in alpha-helical regions. The orientation and location of such helical peptides and proteins in the membrane are key factors determining their function and activity. Here we present a new solution state NMR method for obtaining the orientation of helical peptides in a membrane-mimetic environment (micelle-bound) without any chemical perturbation of the peptide-micelle system. By monitoring proton longitudinal relaxation rates upon addition of the freely water-soluble and inert paramagnetic probe Gd(DTPA-BMA) to an alpha-helical peptide, a wavelike pattern with a periodicity of 3.6 residues per turn is observed. The tilt and azimuth (rotation) angle of the helix determine the shape of this paramagnetic relaxation wave and can be obtained by least-square fitting of measured relaxation enhancements. Results are presented for the 15-residue antimicrobial peptide CM15 which forms an amphipathic helix almost parallel to the surface of the micelle. Thus, a few fast experiments enable the identification of helical regions and determination of the helix orientation within the micelle without the need for covalent modification, isotopic labeling, or sophisticated equipment. This approach opens a path toward the topology determination of alpha-helical membrane-proteins without the need for a complete NOE-based structure determination.     

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.     

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.     

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.     

Rubbing-induced anisotropy of long alkyl side chains at polyimide surfaces

Molecular organization at polyimide surfaces used as alignment layers in liquid crystal displays was investigated using vibrational sum frequency generation (SFG) spectroscopy. We focus on the orientation of the long alkyl side groups at the polymer surface using polarization-selected SFG spectra of the CH(3)- and CH(2)-stretch modes of the side chain. Mechanical rubbing and baking, an accepted industrial procedure used to produce pretilt of the liquid crystal, was found to induce pronounced azimuthal anisotropy in the orientational distribution of the alkyl side chains. Orientational analysis of the SFG vibrational spectra in terms of the azimuthal and tilt angles (in and out of plane, respectively) of the alkyl side chains shows their preferential tilt along the rubbing direction, with the azimuthal distribution narrower for stronger rubbed polymer samples.     

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.     

Unique Vibrational Features as a Direct Probe of Specific Antigen–Antibody Recognition at the Surface of a Solid‐Supported Hybrid Lipid Bilayer

Here, we demonstrate how sum frequency generation (SFG), a vibrational spectroscopy based on a nonlinear three‐photon mixing process, may provide a direct and unique fingerprint of bio‐recognition; This latter can be detected with an intrinsically discriminating unspecific adsorption, thanks to the high sensitivity of the second‐order nonlinear optical (NLO) response to preferential molecular orientation and symmetry properties. As a proof of concept, we have detected the biological event at the solid/liquid interface of a model bio‐active antigen platform, based on a solid‐supported hybrid lipid bilayer (ss‐HLB) of a 2,4‐dinitrophenyl (DNP) lipid, towards a monoclonal mouse anti‐DNP complementary antibody.     

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.     

Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy

In addition to providing a semipermeable barrier that protects a cell from harmful stimuli, lipid membranes occupy a central role in hosting a variety of biological processes, including cellular communications and membrane protein functions. Most importantly, protein-membrane interactions are implicated in a variety of diseases and therefore many analytical techniques were developed to study the basis of these interactions and their influence on the molecular architecture of the cell membrane. In this study, sum frequency generation (SFG) vibrational spectroscopy is used to investigate the spontaneous membrane insertion process of cytochrome b(5) and its mutants. Experimental results show a significant difference in the membrane insertion and orientation properties of these proteins, which can be correlated with their functional differences. In particular, our results correlate the nonfunctional property of a mutant cytochrome b(5) with its inability to insert into the lipid bilayer. The approach reported in this study could be used as a potential rapid screening tool in measuring the topology of membrane proteins as well as interactions of biomolecules with lipid bilayers in situ.     

Label-free chiral detection of melittin binding to a membrane

The study presented here describes an innovative approach for the detection of surface-confined proteins using chiral second harmonic generation (C-SHG). A unique optical geometry has been employed which allows for the separation of the chiral and achiral nonlinear response. By utilizing this optical arrangement, the detection of chirality originating from melittin adsorbed to a planar supported lipid bilayer has been performed for the first time by C-SHG. Melittin binding to the membrane was monitored as a function of bulk concentration through detection of the C-SHG signal. Analysis of the C-SHG adsorption isotherms reveals Frumkin adsorption behavior with a positive interaction energy. The binding constant (Ka) obtained was determined to be (8.3 +/- 1.0) x 105 M-1. The results of these studies have far-reaching implication in the use of C-SHG for the label-free detection of protein association to surfaces and in the analysis of protein interfacial phenomena.     

Mechanisms of the interaction of alpha-helical transmembrane peptides with phospholipid bilayers

The synthetic peptide acetyl-K(2)-G-L(24)-K(2)-A-amide (P(24)) and its analogs have been successfully utilized as models of the hydrophobic transmembrane alpha-helical segments of integral membrane proteins. The central polyleucine region of these peptides was designed to form a maximally stable, very hydrophobic alpha-helix which will partition strongly into the hydrophobic environment of the lipid bilayer core, while the dilysine caps were designed to anchor the ends of these peptides to the polar surface of the lipid bilayer and to inhibit the lateral aggregation of these peptides. Moreover, the normally positively charged N-terminus and the negatively charged C-terminus have both been blocked in order to provide a symmetrical tetracationic peptide, which will more faithfully mimic the transbilayer region of natural membrane proteins and preclude favorable electrostatic interactions. In fact, P(24) adopts a very stable alpha-helical conformation and transbilayer orientation in lipid model membranes. The results of our recent studies of the interaction of this family of alpha-helical transmembrane peptides with phospholipid bilayers are summarized here.