Vibrational spectroscopic studies on fibrinogen adsorption at polystyrene/protein solution interfaces: hydrophobic side chain and secondary structure changes

Structural changes of fibrinogen after adsorption to polystyrene (PS) were examined at the PS/protein solution interface in situ using sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Different behaviors of hydrophobic side chains and secondary structures of adsorbed fibrinogen molecules have been observed. Our results indicate that upon adsorption, the hydrophobic PS surface induces fast structural changes of fibrinogen molecules by aligning some hydrophobic side chains in fibrinogen so that they face to the surface. Such structural changes of fibrinogen hydrophobic side chains are local changes and do not immediately induce significant changes of the protein secondary structures. Our research also shows that the interactions between adsorbed fibrinogen and the PS surface can induce significant changes of protein secondary structures or global conformations which occur on a much longer time scale.     

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.     

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.     

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.     

Infrared-visible sum frequency generation spectroscopic study of molecular orientation at polystyrene/comb-polymer interfaces

Surface-sensitive infrared-visible sum frequency generation spectroscopy (SFG) in total internal reflection geometry has been used to study the structure of poly(vinyl n-octadecyl carbamate-co-vinyl acetate) (PVNODC) or poly(octadecyl acrylate) (PA-18) in contact with a deuterated or hydrogenated polystyrene (dPS or hPS) layer. SFG spectra from the PVNODC (or PA-18)/hPS interface show methyl and methylene peaks corresponding to PVNODC (or PA-18) and phenyl peaks corresponding to the PS. Analysis suggests that the methyl groups are tilted at angles less than 30 degrees with respect to the surface normal. The presence of a strong methylene peak suggests the PVNODC alkyl side chains contain more gauche defects at the PS/PVNODC interface in comparison to PVNODC (or PA-18)/air interfaces. On heating, the SFG intensity from the PS/PA-18 interface drops sharply near the bulk melting temperature (T(m)) of PA-18. Interestingly, a similar drop in SFG signal is also observed for the PS phenyl groups. This demonstrates the ability of the phenyl group at the PS/PA-18 interface to rearrange itself upon the solid-to-liquid transition of the PA-18 alkyl side chain, at a temperature well below the bulk PS glass transition temperature. For PS/PVNODC interfaces, the drop in SFG intensity is gradual and in agreement with the three broad transitions of PVNODC observed in the bulk.     

Diffusion of one or more components of a silane adhesion-promoting mixture into poly(methyl methacrylate)

The surface-sensitive technique of sum frequency generation (SFG) vibrational spectroscopy has been applied to study the buried interfaces between different polymers including deuterated polystyrene (d-PS) and deuterated poly(methyl methacrylate) (d-PMMA) and a two-component silane adhesion-promoting mixture (SAPM) composed of (3-glycidoxypropyl)trimethoxysilane (gamma-GPS) and a methylvinylsiloxanol (MVS). Because of the dissolution of d-PS, no SFG CH stretching signals could be collected from the d-PS/gamma-GPS interface, and SFG signals collected from the d-PS/SAPM interface gradually disappeared over time. SFG results also showed that gamma-GPS can diffuse through the d-PMMA film. The diffusion of gamma-GPS through the d-PMMA film was confirmed by SFG studies on the interface between gamma-GPS and a d-PMMA/PS two-polymer layer system. Initially the SFG signal from the PS layer was detected. However, after gamma-GPS diffused through the d-PMMA film, the PS film was dissolved by the silane, and thus the SFG signal from PS was lost. Similar experiments have been carried out at the interface between the SAPM and the d-PMMA/PS two-polymer layer system and it was found that the diffusion time of the gamma-GPS in the SAPM through the d-PMMA film was significantly longer. These results were much different to those from previous SFG studies on the analogous PET interfaces and appear consistent with differences in solubility parameters calculated for these systems.     

In situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic polystyrene and hydrophilic silica surfaces using quartz crystal microbalance, atomic force microscopy, and sum frequency generation vibrational spectroscopy

The adsorption of a 14-amino acid amphiphilic peptide, LK14, which is composed of leucine (L, nonpolar) and lysine (K, charged), on hydrophobic polystyrene (PS) and hydrophilic silica (SiO2) was investigated in situ by quartz crystal microbalance (QCM), atomic force microscopy (AFM), and sum frequency generation (SFG) vibrational spectroscopy. The LK14 peptide, adsorbed from a pH 7.4 phosphate-buffered saline (PBS) solution, displayed very different coverage, surface roughness and friction, topography, and surface-induced orientation when adsorbed onto PS versus SiO2 surfaces. Real-time QCM adsorption data revealed that the peptide adsorbed onto hydrophobic PS through a fast (t < 2 min) process, while a much slower (t > 30 min) multistep adsorption and rearrangement occurred on the hydrophilic SiO2. AFM measurements showed different surface morphologies and friction coefficients for LK14 adsorbed on the two surfaces. Surface-specific SFG spectra indicate very different ordering of the adsorbed peptide on hydrophobic PS as compared to hydrophilic SiO2. At the LK14 solution/PS interface, CH resonances corresponding to the hydrophobic leucine side chains are evident. Conversely, only NH modes are observed at the peptide solution/SiO2 interface, indicating a different average molecular orientation on this hydrophilic surface. The surface-dependent difference in the molecular-scale peptide interaction at the solution/hydrophobic solid versus solution/hydrophilic solid interfaces (measured by SFG) is manifested as significantly different macromolecular-level adsorption properties on the two surfaces (determined via AFM and QCM experiments).     

Surface structures and properties of polystyrene/poly(methyl methacrylate) blends and copolymers

Sum frequency generation (SFG) vibrational spectroscopy has been applied to study the molecular surface structures of polystyrene (PS)/poly(methyl methacrylate) (PMMA) blends and the copolymer between PS and PMMA (PS-co-PMMA) in air, supplemented by atomic force microscopy (AFM) and contact angle goniometer. Both the blend and the copolymer have equal weight amounts of the two components. SFG results show that both components, PS and PMMA, can segregate to the surface of the blend and the copolymer before annealing, although PMMA has a slightly higher surface tension. Upon annealing both SFG results and contact angle measurements indicate that the PS segregates to the surface of the PS/PMMA blend more but no change occurs on the PS-co-PMMA surface. AFM images show that the copolymer surface is flat but the 1:1 PS/PMMA blend has a rougher surface with island like domains present. The annealing effect on the blend surface morphology has also been investigated. We collected amide SFG signals from interfacial fibrinogen molecules at the copolymer or blend/protein solution interfaces as a function of time. Different time-dependent SFG signal changes have been observed, showing that different surfaces of the blend and the copolymer mediate fibrinogen adsorption behavior differently.     

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.     

The adsorbed conformation of globular proteins at the air/water interface

External reflection FTIR spectroscopy and surface pressure measurements were used to compare conformational changes in the adsorbed structures of three globular proteins at the air/water interface. Of the three proteins studied, lysozyme, bovine serum albumin and beta-lactoglobulin, lysozyme was unique in its behaviour. Lysozyme adsorption was slow, taking approximately 2.5 h to reach a surface pressure plateau (from a 0.07 mM solution), and led to significant structural change. The FTIR spectra revealed that lysozyme formed a highly networked adsorbed layer of unfolded protein with high antiparallel beta-sheet content and that these changes occurred rapidly (within 10 min). This non-native secondary structure is analogous to that of a 3D heat-set protein gel, suggesting that the adsorbed protein formed a highly networked interfacial layer. Albumin and beta-lactoglobulin adsorbed rapidly (reaching a plateau within 10 min) and with little change to their native secondary structure.     

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.     

Conformation-dependent racemization of aspartyl residues in peptides

Biologically uncommon D-aspartyl (D-Asp) residues have been detected in proteins of various tissues of elderly humans. The presence of D-Asp has been explained as a result of the racemization of L-Asp (denoted as Asp) in the protein of inert tissues. We have previously suggested that the racemization of Asp may depend on the conformation of the peptide chain. However, the nature of the peptide conformation that affects the D-Asp formation has not yet been examined. Here we report the kinetics of Asp racemization in two model peptides, (Asp-Leu)(15) and (Leu-Asp-Asp-Leu)(8)-Asp, which form beta-sheet structures and alpha-helical structures, respectively. For the beta-sheet structures, the activation energy of racemization of Asp residues was 27.3 kcal mol(-1), the racemization rate constant at 37 degrees C was 2.14x10(-2) per year and the time required to reach a D/L ratio of 0.99 at 37 degrees C was 122.6 years as estimated from the Arrhenius equation. For the alpha-helical structures, the activation energy of racemization was 18.4 kcal mol(-1), the racemization rate constant 20.02x10(-2) per year and the time 13.1 year. These results suggest that Asp residues inserted in alpha-helical peptides are more sensitive to racemization than Asp residues inserted in peptides adopting beta-sheet structures. The results clearly indicate that the racemization rate of Asp residues in peptides depends on the secondary structure of the host peptide.     

Role of interfacial water on protein adsorption at cross-linked polyethylene oxide interfaces

Sum frequency generation (SFG) vibrational spectroscopy was used to study the structure of water at cross-linked PEO film interfaces in the presence of human serum albumin (HSA) protein. Although PEO is charge neutral, the PEO film/water interface exhibited an SFG signal of water similar to that of a highly charged water/silica interface, signifying the presence of ordered water. Ordered water molecules were observed not only at the water/PEO interface, but also within the PEO film. It indicates that the PEO and water form an ordered hydrogen-bonded network extending from the bulk PEO film into liquid water, which can provide an energy barrier for protein adsorption. Upon exposure to the protein solution, the SFG spectra of water at the water/PEO interface remained nearly unperturbed. For comparison, the SFG spectra of water/silica and water/polystyrene interfaces were also studied with and without HSA in the solution. The SFG spectra of the interfacial water were correlated with the amount of protein adsorbed on the surfaces using fluorescence microscopy, which showed that the amount of protein adsorbed on the PEO film was about 10 times less than that on a polystyrene film and 3 times less than that on silica.     

Molecular‐level structures at poly(4‐vinyl pyridine)/acid interfaces probed by nonlinear vibrational spectroscopy

The molecular structures of the interfaces between a solid poly(4‐vinyl pyridine) (P4VP) surface and poly(acrylic acid) (PAA) as well as hydrochloric acid (HCl) solutions were probed using sum frequency generation (SFG) vibrational spectroscopy in situ in real time. Spectroscopic results clearly reveal that the PAA molecules are adsorbed onto the P4VP surface via hydrogen bonding at the P4VP/PAA solution interface while the P4VP surface is protonated at the P4VP/HCl solution interface. Consequently, the water molecules near the interfaces are strongly perturbed by these two interactions, exhibiting different orderings at the two interfaces. This work clearly demonstrates the power of studying the interfacial molecular‐level structures via nonlinear vibrational spectroscopy when molecular adsorption happens at the solid–liquid interface and paves a way for our future study on tracing the adsorption dynamics of polymer chains onto solid surfaces. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016 , 54, 848–852     

Conformation study of HA(306-318) antigenic peptide of the haemagglutinin influenza virus protein

Several HLA-DR alleles present the immunodominant HA(306-318) peptide of haemagglutinin of the influenza virus to T cells. NMR data of the peptide in various water solutions exclude any alpha-helix or turn conformations. Circular dichroism and Fourier transform infrared spectroscopies indicate an estimated beta-extended structure in water of 31% and 28%, respectively, with spectra shape similar to the ones observed for beta-sheet containing proteins. The H/D amide exchange suggests a stable length-dependent interchain hydrogen-bonding. The partially beta-extended conformation of HA(306-318) in solution might be close to the one found in HA(306-318)-HLA-DR1 complex. These results suggest different interconverting extended conformations of HA(306-318), depending on the microenvironment of the solution medium. This flexibility emphasizes the ability of some peptides to fit more easily the binding site of several HLA-DR molecules. Similar results were obtained on the HIV P25(263-277) peptide which has been previously shown to be a good DR1 binder. From a vibrational point of view, infrared Amide I frequencies of secondary structures in peptides were ascertained. As previously demonstrated for proteins in solution, Fourier transform infrared and circular dichroism spectroscopies appear to be valuable tools for conformational properties of peptides. Their use may contribute to the detection of peptide conformation-binding relationship which has to be further tested by biochemical and biological studies.     

Demonstrating the feasibility of monitoring the molecular-level structures of moving polymer/silane interfaces during silane diffusion using SFG

In this paper, the feasibility of monitoring molecular structures at a moving polymer/liquid interface by sum frequency generation (SFG) vibrational spectroscopy has been demonstrated. N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane (AATM, NH2(CH2)2NH(CH2)3Si(OCH3)3) has been brought into contact with a deuterated poly(methyl methacrylate) (d-PMMA) film, and the interfacial silane structure has been monitored using SFG. Upon initial contact, the SFG spectra can be detected, but as time progresses, the spectral intensity changes and finally disappears. Additional experiments indicate that these silane molecules can diffuse into the polymer film and the detected SFG signals are actually from the moving polymer/silane interface. Our results show that the molecular order of the polymer/silane interface exists during the entire diffusion process and is lost when the silane molecules traverse through the thickness of the d-PMMA film. The loss of the SFG signal is due to the formation of a new disordered substrate/silane interface, which contributes no detectable SFG signal. The kinetics of the diffusion of the silane into the polymer have been deduced from the time-dependent SFG signals detected from the AATM molecules as they diffuse through polymer films of different thickness.     

Following the Azide‐Alkyne Cycloaddition at the Silica/Solvent Interface with Sum Frequency Generation

The Cu^I‐catalyzed 1,3‐dipolar azide‐alkyne cycloaddition (CuAAC) has arisen as one of the most useful chemical transformations for introducing complexity onto surfaces and materials owing to its functional‐group tolerance and high yield. However, methods for monitoring such reactions in situ at the widely used silica/solvent interface are hampered by challenges associated with probing such buried interfaces. Using the surface‐specific technique broadband sum frequency generation (SFG), we monitored the reaction of a benzyl azide monolayer in real time at the silica/methanol interface. A strong peak at 2096 cm^?1 assigned to the azides was observed for the first time by SFG. Using a cyano‐substituted alkyne, the decrease of the azide peak and the increase of the cyano peak (2234 cm^?1) were probed simultaneously. From the kinetic analysis, the reaction order with respect to copper was determined to be 2.1, suggesting that CuAAC on the surface follows a similar mechanism as in solution.     

隐藏高分子界面及生物界面分子结构的和频振动光谱研究

界面的分子结构决定界面的性质.为了以优化界面的结构来改进材料的性质,原位实时地研究界面的分子结构是很重要的.近年来和频振动光谱已发展成为一个很有效及独特的手段来研究隐藏界面的分子结构,例如液/液界面、固/液界面及固/固界面等.这篇综述讨论了和频振动光谱在研究高分子界面及生物界面等复杂界面的分子结构上的应用.具体说来,本文论述了高分子表面在水里的分子结构变化,高分子及模型粘合促进剂硅烷在界面相互作用的分子机理和隐藏的高分子/高分子及高分子/金属界面的结构.另外,此文还将介绍不同二级结构的多肽及几个有代表性的蛋白分子在界面的结构.界面在诸如化学、生物、物理、材料科学及工程和纳米技术等许多领域都很重要.发展一个独特的能原位研究隐藏界面的分子结构的技术会有力地促进这些领域的研究及跨学科研究的发展.     

Reinvestigation on the buildup mechanism of alternate multilayers consisting of poly(L-glutamic acid) and poly(L-, D-, and DL-lysines)

The buildup mechanism of polypeptide multilayers prepared by the layer-by-layer deposition of a polyanion (poly(L-glutamic acid) (PGA)) and polycations (poly(L-lysine) (PLL), poly(D-lysine) (PDL), and copoly(DL-lysine)(PDLL)) was reinvestigated by using in situ ATR-IR spectroscopy. A difference spectral technique applied to analyze the spectra indicated that the deposition of both the PGA and PLL (PDL) layers accompanies the formation of secondary structures consisting mainly of the antiparallel pleated sheet (the beta-sheet) structure, and that the formation of the beta-sheet structure cannot always be explained in terms of polyanion/polycation complex formation or charge compensation between the polyanion and polycations, although it has been considered as a major process in the multilayer buildup process. Instead, the present paper proposes the following mechanism. During the deposition of the polyelectrolyte, a small amount of the beta-sheet structures are produced at the interface as a result of charge compensation between a polyelectrolyte and an oppositely charged polyelectrolyte in the multilayer. The beta-sheets act as nuclei from which further propagation of the structure takes place at the solution/multilayer interfaces. The driving force of the buildup process in the new mechanism is a kinetically favorable insolubilization of each polyelectrolyte in solution at the interfaces.     

Microscopic Investigation of Ethylene Carbonate Interface: A Molecular Dynamics and Vibrational Spectroscopic Study

Ethylene carbonate(EC) liquid and its vapor-liquid interface were investigated using a combination of molecular dynamics(MD)simulation and vibrational IR, Raman and sum frequency generation(SFG)spectroscopies. The MD simulation was performed with a flexible and polarizable model of the EC molecule newly developed for the computation of vibrational spectra. The internal vibration of the model was described on the basis of the harmonic couplings of vibrational modes, including the anharmonicity and Fermi resonance coupling of C=O stretching. The polarizable model was represented by the charge response kernel(CRK),which is based on ab initio molecular orbital calculations and can be readily applied to other systems. The flexible and polarizable model can also accurately reproduce the structural and thermodynamic properties of EC liquid. Meanwhile, a comprehensive set of vibrational spectra of EC liquid, including the IR and Raman spectra of the bulk liquid as well as the SFG spectra of the liquid interface, were experimentally measured and reported. The set of experimental vibrational spectra provided valuable information for validating the model, and the MD simulation using the model comprehensively elucidates the observed vibrational IR, Raman, and SFG spectra of EC liquid. Further MD analysis of the interface region revealed that EC molecules tend to orientate themselves with the C=O bond parallel to the interface. The MD simulation explains the positive Im[χ~((2))](ssp) band of the C=O stretching region in the SFG spectrum in terms of the preferential orientation of EC molecules at the interface. This work also elucidates the distinct lineshapes of the C=O stretching band in the IR, Raman, and SFG spectra. The lineshapes of the C=O band are split by the Fermi resonance of the C=O fundamental and the overtone of skeletal stretching. The Fermi resonance of C=O stretching was fully analyzed using the empirical potential parameter shift analysis(EPSA) method. The apparently different lineshapes of the C=O stretching band in the IR, Raman, and SFG spectra were attributed to the frequency shift of the C=O fundamental in different solvation environments in the bulk liquid and at the interface. This work proposes a systematic procedure for investigating the interface structure and SFG spectra, including general modeling procedure based on ab initio calculations, validation of the model using available experimental data, and simultaneous analysis of molecular orientation and SFG spectra through MD trajectories. The proposed procedure provides microscopic information on the EC interface in this study, and can be further applied to investigate other interface systems, such as liquid-liquid and solid-liquid interfaces.