Fermi Resonant Interaction of the Tailed Methyl Groups of Langmuir Monolayer at the Air/Water Interface during Phase Transition

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-<i>sn</i>-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 (<i>R</i>₁) between Fermi resonance and symmetric stretching mode of the tailed methyl groups sharply decreases during the phase transition. The sharp drop of <i>R</i>₁ 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 <i>R</i>₁. However, such phenomena are not observed at the air/KCl solution (0.3M) interface

Molecular-Level Insights into N–N π‑Bond Rotation in the pH-Induced Interfacial Isomerization of 5‑Octadecyloxy-2-(2-pyridylazo)phenol Monolayer Investigated by Sum Frequency Generation Vibrational Spectroscopy

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 (<i>A</i>) 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

Observing Peptide-Induced Lipid Accumulation in a Single-Component Zwitterionic Lipid Bilayer

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

Specific Ion Effects on Protein Thermal Aggregation from Dilute Solutions to Crowded Environments

We have investigated specific ion effects on protein thermal aggregation from dilute solutions to crowded environments. Ovalbumin and poly­(ethylene glycol) have been employed as the model protein and crowding agent, respectively. Our studies demonstrate that the rate-limiting step of ovalbumin thermal aggregation is changed from the aggregation of unfolded protein molecules to the unfolding of the protein molecules, when the solution conditions are varied from a dilute solution to a crowded environment. The specific ion effects acting on the thermal aggregation of ovalbumin generated by kosmotropic and chaotropic ions are different. The thermal aggregation of ovalbumin molecules is promoted by kosmotropic anions in dilute solutions via an increase in protein hydrophobic interactions. In contrast, ovalbumin thermal aggregation is facilitated by chaotropic ions in crowded environments through accelerated unfolding of protein molecules. Therefore, there are distinct mechanisms causing the ion specificities of protein thermal aggregation between dilute solutions and crowded environments. The ion specificities are dominated by ion-specific hydrophobic interactions between protein molecules and ion-specific unfolding of protein molecules in dilute solutions and crowded environments, respectively

Reversible Activation of pH-Responsive Cell-Penetrating Peptides in Model Cell Membrane Relies on the Nature of Lipid

The pH response of pH-responsive cell-penetrating peptides in cell membrane is directly associated with many potential applications and cell activities such as drug delivery, membrane fusion, and protein folding, but it is still poorly understood. In this study, we used GALA as a model and applied sum frequency generation vibrational spectroscopy to systematically investigate the pH response of GALA in lipid bilayers with different hydrophobic length and lipid head groups. We determined the GALA structures in lipid bilayers by combining second-ordered amide I and amide III spectral signals, which can accurately differentiate the loop and α-helical structures at the interface. It is found that GALA can insert into fluid-phase lipid bilayers even at neutral pH, while lies down on the gel-phase lipid bilayer surface. Under acidic conditions, GALA inserts into both fluid-phase and gel-phase lipid bilayers. GALA adopts a mixed loop and α-helical structures in lipid bilayers. Besides, the reversible activation of GALA in lipid bilayers depends on the nature of lipid. After membrane insertion, GALA exits from the negative phosphoglycerol and positive ethylphosphocholine lipid bilayers at neutral pH, while it does not move out from the zwitterionic phosphocholine lipid bilayers. These findings will help us to understand how to enhance the efficacy of drug/gene delivery in cell membrane

Specific Ion Interaction Dominates over Hydrophobic Matching Effects in Peptide–Lipid Bilayer Interactions: The Case of Short Peptide

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

Transport and Organization of Cholesterol in a Planar Solid-Supported Lipid Bilayer Depend on the Phospholipid Flip-Flop Rate

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

Intermolecular Interactions at the Interface Quantified by Surface-Sensitive Second-Order Fermi Resonant Signals

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 <i>in situ</i> 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 <i>in</i> <i>situ</i> and in real time

Phosphate Ions Promoting Association between Peptide and Modeling Cell Membrane Revealed by Sum Frequency Generation Vibrational Spectroscopy

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

Accurate Determination of Interfacial Protein Secondary Structure by Combining Interfacial-Sensitive Amide I and Amide III Spectral Signals

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