Enhanced Photostability and Photoactivity of Ruthenium Polypyridyl-Based Photocatalysts by Covalently Anchoring Onto Reduced Graphene Oxide.

Recentstudies toward finding more efficient ruthenium metalloligands for photocatalysis applications have shown that the derivatives of the linear [Ru(dqp)2]2+ (dqp: 2,6-di(quinolin-8-yl)-pyridine) complexes hold significant promise due to their extended emission lifetime in the µs time scale while retaining comparable redox potential, extinction coefficients, and absorption profile in the visible region to [Ru(bpy)3]2+ (bpy: 2,2'-bipyridine) and [Ru(tpy)2]2+ (tpy: 2,2':6',2″-terpyridine) complexes. Nevertheless, its photostability in aqueous solution needs to be improved for its widespread use in photocatalysis. Carbon-based supports have arisen as potential solutions for improving photostability and photocatalytic activity, yet their effect greatly depends on the interaction of the metal complex with the support. Herein, we present a strategy for obtaining Ru-polypyridyl complexes covalently linked to aminated reduced graphene oxide (rGO) to generate novel materials with long-term photostability and increased photoactivity. Specifically, the hybrid Ru(dqp)@rGO system has shown excellent photostable behavior during 24 h of continual irradiation, with an enhancement of 10 and 15% of photocatalytic dye degradation in comparison with [Ru(dqp)2]2+ and Ru(tpy)@rGO, respectively, as well as remarkable recyclability. The presented strategy corroborates the potential of [Ru(dqp)2]2+ as an interesting photoactive molecule to produce more advantageous light-active materials by covalent attachment onto carbon-based supports.

Evaluation of the passive permeability of antidepressants through pore-suspended lipid bilayer.

HYPOTHESIS: The antidepressant drug imipramine, and its metabolite desipramine show different extents of interaction with, and passive permeation through, cellular membrane models, with the effects depending on the membrane composition. Through multimodal interrogation, we can observe that the drugs have a direct impact on the physicochemical properties of the membrane, that may play a role in their pharmacokinetics. EXPERIMENTS: Microcavity pore-suspended lipid bilayers (MSLBs) of four different compositions, each with a different headgroup charge namely; zwitterionic dioleoylphosphatidylcholine (DOPC), mixed DOPC and negatively charged dioleoylphosphatidylglycerol (DOPG) (3:1), mixed DOPC and positively charged dioleoyltrimethylammoniumpropane (DOTAP) (3:1), and with increasing complex composition mimicking blood-brain-barrier (BBB) were prepared on gold and polydimethylsiloxane (PDMS) substrates using a Langmuir-Blodgett-vesicle fusion method. The molecular interaction and permeation of antidepressants, imipramine, and its metabolite desipramine with the lipid bilayers were evaluated using highly sensitive label-free electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS). Drug-induced membrane packing/fluidity alterations were assessed using fluorescence lifetime imaging (FLIM) and fluorescence lifetime correlation spectroscopy (FLCS) of MSLB over microfluidic PDMS array. FINDINGS: Using EIS to evaluate in real-time membrane admittance changes, we found that imipramine greatly increases the ion permeability of negatively charged DOPC:DOPG (3:1) membranes. The effect was observed also at neutral (DOPC) and to a lesser extent at positively charged DOPC:DOTAP(3:1) membranes. In contrast, desipramine had a much weaker impact on ion permeability across all bilayer compositions. Temporal capacitance data show that desipramine intercalates at negatively charged membrane thereby increasing the thickness of the membrane. The overall kinetics of the imipramine permeation is higher than that of desipramine. This was confirmed using SERS, which also provides an evaluation of drug passive permeation based on arrival time across the membrane. Using FLCS, we found that imipramine increases the lipid membrane fluidity, whereas desipramine lowers it, with the exception of the negatively charged membrane. A translocation rate pharmacokinetics model was established for the first time at the MSLB platform by real-time monitoring of the variation in membrane resistance of pristine DOPC and blood-brain-barrier (BBB) membrane.

Leaflet by Leaflet Synergistic Effects of Antimicrobial Peptides on Bacterial and Mammalian Membrane Models.

Antimicrobial peptides (AMPs) offer significant hope in the fight against antibiotic resistance. Operating via a mechanism different from that of antibiotics, they target the microbial membrane and ideally should damage it without impacting mammalian cells. Here, the interactions of two AMPs, magainin 2 and PGLa, and their synergistic effects on bacterial and mammalian membrane models were studied using electrochemical impedance spectroscopy, atomic force microscopy (AFM), and fluorescence correlation spectroscopy. Toroidal pore formation was observed by AFM when the two AMPs were combined, while individually AMP effects were confined to the exterior leaflet of the bacterial membrane analogue. Using microcavity-supported lipid bilayers, the diffusivity of each bilayer leaflet could be studied independently, and we observed that combined, the AMPs penetrate both leaflets of the bacterial model but individually each peptide had a limited impact on the proximal leaflet of the bacterial model. The impact of AMPs on a ternary, mammalian mimetic membrane was much weaker.

Role of phosphatidylserine in amyloid-beta oligomerization at asymmetric phospholipid bilayers.

Amyloid-beta (Aß1-42) aggregation triggers neurotoxicity and is linked to Alzheimer's disease. Aß1-42 oligomers, rather than extended fibrils, adhere to the cell membrane, causing cell death. Phosphatidylserine (PS), an anionic phospholipid, is prevalent in neuronal membranes (< 20 molar percentage) and, while isolated to the cytoplasmic leaflet of the membrane in healthy cells, its exposure in apoptotic cells and migration to exoplasmic leaflet is triggered by oxidative damage to the membrane. It is widely believed that PS plays a crucial role in the Aß peptide interaction in the membranes of neuronal cells. However, due to the complexity of the cell membrane, it can be challenging to address molecular level understanding of the PS-Aß binding and oligomerization processes. Herein, we use microcavity supported lipid bilayers (MSLBs) to analyse PS and Aß1-42 binding, oligomer formation, and membrane damage. MSLBs are a useful model to evaluate protein-membrane interactions because of their cell-like dual aspect fluidity, their addressability and compositional versatility. We used electrochemical impedance spectroscopy (EIS) and confocal fluorescence microscopy to compare the impact of Aß1-42 on simple zwitterioinic membrane, dioleoylphosphatidylcholine (DOPC), with MSLBs comprised of transversally asymmetric binary DOPC and dioleoylphosphatidylserine (DOPS). Monomeric Aß1-42 adsorbs weakly to the pristine zwitterionic DOPC membrane without aggregation. Using a membrane integrity test, with pyranine trapped within the cavities beneath the membrane, Aß1-42 exposure did not result in pyranine leakage, indicating that DOPC membranes were intact. When 10 mol% DOPS was doped asymmetrically into the membrane's outer leaflet, oligomerization of Aß1-42 monomer was evident in EIS and atomic force microscopy (AFM), and confocal imaging revealed that membrane damage, resulted in extensive pyranine leakage from the pores. The effects were time, and DOPS and Aß1-42 concentration-dependent. Membrane pore formation was visible within 30 minutes, and oligomerization, membrane-oligomer multilayer, and Aß1-42 fibril formation evident over 3 to 18 hours. In asymmetric membranes with DOPS localized to the lower leaflet, optothermally (laser induced) damage increased local DOPS concentrations at the distal leaflet, promoting Aß1-42 aggregation.

Galectin-3 Binding to α5ß1 Integrin in Pore Suspended Biomembranes.

Galectin-3 (Gal3) is a ß-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5ß1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5ß1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane's admittance and fluidity, as well as integrin's lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan-Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5ß1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3-α5ß1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

A cell free biomembrane platform for multimodal study of influenza virus hemagglutinin and for evaluation of entry-inhibitors against hemagglutinin.

Seasonal periodic pandemics and epidemics caused by Influenza A viruses (IAVs) are associated with high morbidity and mortality worldwide. They are frequent and unpredictable in severity so there is a need for biophysical platforms that can be used to provide both mechanistic insights into influenza virulence and its potential treatment by anti-IAV agents. Host membrane viral association through the glycoprotein hemagglutinin (HA) of IAVs is one of the primary steps in infection. HA is thus a potential target for drug discovery and development against influenza. Deconvolution of the multivalent interactions of HA at the interfaces of the host cell membrane can help unravel therapeutic targets. In this contribution, we reported the effect of a multivalent HA glycoprotein association on various glycosphingolipid receptors (GD1a, GM3, GM1) doped asymmetrically into an artificial host membrane spanned across an aqueous filled microcavity array. The extent of HA association and its impact on membrane resistance, capacitance, and diffusivity was measured using highly sensitive electrochemical impedance spectroscopy (EIS) and fluorescence lifetime correlation spectroscopy (FLCS). Furthermore, we investigated the inhibition of the influenza HA glycoprotein association with the host mimetic surface by natural and synthetic sialic acid-based inhibitors (sialic acid, Siaα2,3-GalOMe, FB127, 3-sialyl lactose) using electrochemical impedance spectroscopy and observe that while all inhibit, they do not prevent host binding. Overall, the work demonstrates the platform provides a label-free screening platform for the biophysical evaluation of new inhibitors in the development of potential therapeutics for IAV infection prevention and treatment.

Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers.

Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine's influence on the cell membrane, including permeability, binding, and rearrangement at the molecular level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as in vitro models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid composition on the membrane interactions of quinacrine. Using electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Our EIS data reveal that the drug is permeable through ternary DOPC:SM:Chol and DOPC-only bilayer compositions. In contrast, the binary cholesterol/DOPC membrane arrested permeation, yet the drug binds or intercalates at this membrane as reflected by an increase in membrane impedance. SERS supported the EIS data, which was utilized to gain structural insights into the drug-membrane interaction. Our SERS data also provides a simple but powerful label-free assessment of drug permeation because a significant SERS enhancement of the drug's Raman signature was observed only if the drug accessed the plasmonic interior of the pore cavity passing through the membrane. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophysical insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug-membrane interactions, making it a powerful tool for in vitro drug screening.

Microcavity array supported lipid bilayer models of ganglioside - influenza hemagglutinin1 binding.

The binding of influenza receptor (HA1) to membranes containing different glycosphingolipid receptors was investigated at Microcavity Supported Lipid Bilayers (MSLBs). We observed that HA1 preferentially binds to GD1a but the diffusion coefficient of the associated complex at lipid bilayer is approximately double that of the complexes formed by HA1 GM1 or GM3.

Dimethylaniline functionalised pyrene fluorophores; dual colour pH switching in solution and self-assembled monolayers.

A pyrene charge transfer fluorophore with three ionizable N,N-dimethylaniline moieities was explored as an interfacial pH switch. The parent carboxylate compound and the thiolated derivative were shown by spectroscopy combined with DFT calculation to be successively and reversibly protonated. Protonation leads to progressive decrease of intensity of the 550 nm centered N,N-dimethylaniline to pyrene charge transfer emission which on protonation of the third site, leads to extinction of this transition and evolution of an intense blue (450 nm) pyrene-centered emission. Concomitant loss of the charge transfer absorbance was observed and the changes are reversed on neutralization of pH. A self-assembled monolayer of the thiolated derivative was prepared on gold and found from voltammetry of ferricyanide/ferrocyanide probe to form close packed monolayers. The probe voltammetry, label-free electrochemical impedance spectroscopy of the film was monitored as a function of pH and progressive, but reversible protonation steps were reflected in decreasing film resistance. The Stokes shift of the probe prevents self-quenching so a broad, charge transfer fluorescence centered around 540 nm was recorded for the self-assembled monolayer where as per solution, progressive and reversible reduction in intensity was observed. The facile assembly, impedance and optical switching make these materials potentially interesting as on-off or two colour on-off-on fluorescence switches with potential applications in logic gates or in responsive surface applications.

Microcavity-Supported Lipid Bilayers; Evaluation of Drug-Lipid Membrane Interactions by Electrochemical Impedance and Fluorescence Correlation Spectroscopy.

Many drugs have intracellular or membrane-associated targets, thus understanding their interaction with the cell membrane is of value in drug development. Cell-free tools used to predict membrane interactions should replicate the molecular organization of the membrane. Microcavity array-supported lipid bilayer (MSLB) platforms are versatile biophysical models of the cell membrane that combine liposome-like membrane fluidity with stability and addressability. We used an MSLB herein to interrogate drug-membrane interactions across seven drugs from different classes, including nonsteroidal anti-inflammatories: ibuprofen (Ibu) and diclofenac (Dic); antibiotics: rifampicin (Rif), levofloxacin (Levo), and pefloxacin (Pef); and bisphosphonates: alendronate (Ale) and clodronate (Clo). Fluorescence lifetime correlation spectroscopy (FLCS) and electrochemical impedance spectroscopy (EIS) were used to evaluate the impact of drug on 1,2-dioleyl- sn-glycerophosphocholine and binary bilayers over physiologically relevant drug concentrations. Although FLCS data revealed Ibu, Levo, Pef, Ale, and Clo had no impact on lipid lateral mobility, EIS, which is more sensitive to membrane structural change, indicated modest but significant decreases to membrane resistivity consistent with adsorption but weak penetration of drugs at the membrane. Ale and Clo, evaluated at pH 5.25, did not impact the impedance of the membrane except at concentrations exceeding 4 mM. Conversely, Dic and Rif dramatically altered bilayer fluidity, suggesting their translocation through the bilayer, and EIS data showed that resistivity of the membrane decreased substantially with increasing drug concentration. Capacitance changes to the bilayer in most cases were insignificant. Using a Langmuir-Freundlich model to fit the EIS data, we propose Rsat as an empirical value that reflects permeation. Overall, the data indicate that Ibu, Levo, and Pef adsorb at the interface of the lipid membrane but Dic and Rif interact strongly, permeating the membrane core modifying the water/ion permeability of the bilayer structure. These observations are discussed in the context of previously reported data on drug permeability and log P.

Macromolecular inversion-driven polymer insertion into model lipid bilayer membranes.

Macromolecules of amphiphilic invertible polymers (AIPs) are capable of self-assembly into micellar assemblies of various morphologies in solvents of different polarities. The micellar assemblies in aqueous media are capable of encapsulating poorly aqueous soluble cargo and can undergo inverse conformational change and cargo release in contact with non-polar media, including potentially, cell membranes. Thus, invertible micellar assemblies have significant potential in drug delivery and related domains. However, to date there have been few investigations into their interactions with lipid membranes. Herein, we investigate the interactions of three recently developed AIPs of varying hydrophobicity/hydrophilicity balance with a highly fluidic microcavity supported 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer. We combined electrochemical impedance spectroscopy (EIS) with fluorescence correlation spectroscopy (FCS) to understand how the AIP micellar assemblies impacted bilayer permeability and fluidity respectively, across polymer concentrations above and below their critical micelle concentrations (cmcs). At concentration as above their cmcs, all of the AIPs explored increased permeability and decreased the fluidity of the lipid membrane. The extent of impact depended on the hydrophobicity of the AIP. PEG600-PTHF650, the most hydrophobic of the polymers, synthesized from PEG (molecular weight 600 g/mol) and PTHF (molecular weight 650 g/mol) exerted the greatest influence on the bilayer's physical properties and fluorescence imaging and correlation data indicate that PEG600-PTHF650 micelles loaded with BODIPY probes adsorb and invert at the lipid membrane with release of cargo into the bilayer. This study should help inform future advancement of AIPs for membrane molecular delivery.

Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition.

Microcavity-supported lipid bilayers (MSLBs) are contact-free membranes suspended across aqueous-filled pores that maintain the lipid bilayer in a highly fluidic state, free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer a significant opportunity to model membrane asymmetry, protein-membrane interactions, and aggregation at the membrane interface. Herein we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity, and cholera toxin-ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phases, and ternary phase-separated membrane compositions. Fluorescence lifetime correlation spectroscopy was used to determine the lateral mobility of the lipid and the protein, and electrochemical impedance spectroscopy enabled the detection of the protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have a profound impact on membrane electrochemical resistance, where the resistance of a ternary symmetric phase-separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition in the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coefficient for MSLBs was observed to be 2.5 times faster for asymmetric MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that the interplay of lipid packing across both membrane leaflets and the concentration of GM1 both affect the extent of cholera toxin aggregation and the consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asymmetric, MSLBs offer the opportunity to build greater biomimicry into biophysical models, and the approach described demonstrates the value of MSLBs in studying aggregation and the membrane-associated multivalent interactions prevalent in many carbohydrate-mediated processes.

Pathways for creation and annihilation of nanoscale biomembrane domains reveal alpha and beta-toxin nanopore formation processes.

Raft-like functional domains with putative sizes of 20-200 nm and which are evolving dynamically are believed to be the most crucial regions in cellular membranes which determine cell signaling and various functions of cells. While the actual sizes of these domains are believed to vary from cell to cell no direct determination of their sizes and their evolution when cells interact with external agents like toxins and relevant biomolecules exists. Here, we report the first direct determination of the size of these nanoscale regions in model raft-forming biomembranes using the method of super-resolution stimulated emission depletion nanoscopy coupled with fluorescence correlation spectroscopy (STED-FCS). We also show that the various pathways for creation and destruction of such nanoscale membrane regions due to interaction with prototypical α and ß nanopore-forming toxins, can reveal the nature of the respective pore formation processes. The methodology, in turn, establishes a new nano-biotechnological protocol which could be very useful in preventing their cytotoxic effects in particular but also enable microscopic understanding of biomolecule-cell membrane interactions in general.

Unraveling complex nanoscale lipid dynamics in simple model biomembranes: Insights from fluorescence correlation spectroscopy in super-resolution stimulated emission depletion mode.

Dynamic heterogeneity (DH) at nanoscale due to lipid-lipid and/or lipid-protein interactions in cell membranes plays a crucial role in determining a broad range of important cell functions. In cell membranes, the dimensions of these nanodomains have been postulated to be in the order of 10's of nm and transient in nature. While the structural features of membranes have been studied in detail, little is known about their dynamical characteristics due to paucity of techniques which can probe nanoscale phenomena with simultaneous high temporal resolution. A combination of super-resolution stimulated emission depletion (STED) and fluorescence correlation spectroscopy (FCS) technique can overcome this limitation and provide information about the nanoscale dynamic heterogeneity in cell membranes. Using STED-FCS and FCS diffusion law, we provide an understanding of how nanoscale dynamically organizing lipid platforms can emerge in minimal system of model biomembranes. To illustrate the utility of the technique we have chosen cholesterol containing supported lipid bilayers and demonstrated the role of cholesterol concentration and/or added pore-forming protein, Listeriolysin O (LLO) in determining onset of lipid DH. In addition we have also looked at multi-component lipid bilayers with and without cholesterol to infer about the role of phospholipid and cholesterol composition on lipid dynamics. These results on simple biomimetic systems provide insights into fundamental pathways for the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events and depict the significant contribution that STED-FCS can make in resolving several outstanding issues in membrane biology.

Complex dynamics at the nanoscale in simple biomembranes.

Nature is known to engineer complex compositional and dynamical platforms in biological membranes. Understanding this complex landscape requires techniques to simultaneously detect membrane re-organization and dynamics at the nanoscale. Using super-resolution stimulated emission depletion (STED) microscopy coupled with fluorescence correlation spectroscopy (FCS), we reveal direct experimental evidence of dynamic heterogeneity at the nanoscale in binary phospholipid-cholesterol bilayers. Domain formation on the length scale of ~200-600 nm due to local cholesterol compositional heterogeneity is found to be more prominent at high cholesterol content giving rise to distinct intra-domain lipid dynamics. STED-FCS reveals unique dynamical crossover phenomena at length scales of ~100-150 nm within each of these macroscopic regions. The extent of dynamic heterogeneity due to intra-domain hindered lipid diffusion as reflected from the crossover length scale, is driven by cholesterol packing and organization, uniquely influenced by phospholipid type. These results on simple binary model bilayer systems provide novel insights into pathways leading to the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events.

Regio-selective lipase catalyzed hydrolysis of oxanorbornane-based sugar-like amphiphiles at air-water interface: A polarized FT-IRRAS study.

Interfacial hydrolysis of oxanorbornane-based amphiphile (Triol C16) by Candida rugosa lipase was investigated using real-time polarized Fourier transform-infrared reflection absorption spectroscopy (FT-IRRAS). The kinetics of hydrolysis was studied by analyzing the ester carbonyl ν(CO) stretching vibration band across the two dimensional (2D) array of molecules at the confined interface. In particular, we demonstrate Triol C16 to form Michaelis-Menten type complex, like that of lipid-substrate analogues, where the Triol C16 head group remained accessible to the catalytic triad of the lipase. The enzyme-induced selective cleavage of the ester bond was spectroscopically monitored by the disappearance of the intense ν(CO) resonance at 1736cm-1. Consequently, the in situ spectroscopic measurements evidenced selective ester hydrolysis of Triol C16 yielding Tetrol C2OH and Palmitic acid, which remained predominantly in the undissociated form at the interface. The conformation sensitive amide I (majorly ν(CO)) and the interfacial water reorganization suggested 2D ordering of the enzyme molecules following which interfacial reactions were employed towards probing the enzyme kinetics at the air/water interface. The investigation demonstrated further the potential of IRRAS spectroscopy for real-time monitoring the hydrolytic product formation and selectivity at biomimetic interfaces.

Nanoscale dynamics of phospholipids reveals an optimal assembly mechanism of pore-forming proteins in bilayer membranes.

Cell membranes are believed to be highly complex dynamical systems having compositional heterogeneity involving several types of lipids and proteins as the major constituents. This dynamical and compositional heterogeneity is suggested to be critical to the maintenance of active functionality and response to chemical, mechanical, electrical and thermal stresses. However, delineating the various factors responsible for the spatio-temporal response of actual cell membranes to stresses can be quite challenging. In this work we show how biomimetic phospholipid bilayer membranes with variable lipid fluidity determine the optimal assembly mechanism of the pore-forming protein, listeriolysin O (LLO), belonging to the class of cholesterol dependent cytolysins (CDCs). By combining atomic force microscopy (AFM) and super-resolution stimulated emission depletion (STED) microscopy imaging on model membranes, we show that pores formed by LLO in supported lipid bilayers can have variable conformation and morphology depending on the fluidity of the bilayer. At a fixed cholesterol concentration, pores formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes were larger, flexible and more prone to coalescence when compared with the smaller and more compact pores formed in the lower fluidity 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. In contrast, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes did not show any evidence of pore formation. Fluorescence correlation spectroscopy (FCS) in STED mode revealed the appearance of a length scale, ξ, below which lipid dynamics, under the influence of LLO protein binding and assembly, becomes anomalous. Interestingly, the magnitude of ξ is found to correlate with both lipid fluidity and pore dimensions (and flexibility) in DOPC and POPC bilayers. However this length scale dependent crossover, signalling the onset of anomalous diffusion, was not observed in DMPC bilayers. Our study highlights the subtle interplay of lipid membrane mediated protein assembly and lipid fluidity in determining proteo-lipidic complexes formed in biomembranes and the significant insight that STED microscopy provides in unraveling critical aspects of nanoscale membrane biophysics.

Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.

Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.

Structure and dynamics of H2O vis-á-vis phenylalanine recognition at a DPPC lipid membrane via interfacial H-bond types: insights from polarized FT-IRRAS and ADMP simulations.

Preferential and enantioselective interactions of L-/D-Phenylalanine (L-Phe and D-Phe) and butoxycarbonyl-protected L-/D-Phenylalanine (LPA and DPA) as guest with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (L-DPPC) as host were tapped by using real time Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS). Polarization-modulated FT-IRRAS of DPPC monolayers above the phenylalanine modified subphases depicted fine structure/conformation differences under considerations of controlled 2D surface pressure. Selective molecular recognition of D-enantiomer over L-enantiomer driven by the DPPC head group via H-bonding and electrostatic interactions was evident spectroscopically. Accordingly, binding constants (K) of 145, 346, 28, and 56 M(-1) for LPA, DPA, L-Phe, and D-Phe, respectively, were estimated. The real time FT-IRRAS water bands were strictly conformation sensitive. The effect of micro-solvation on the structure and stability of the 1:1 diastereomeric L-lipid⋯, LPA/DPA and L-lipid⋯, (L/D)-Phe adducts was investigated with the aid of Atom-centered Density Matrix Propagation (ADMP), a first principle quantum mechanical molecular dynamics approach. The phosphodiester fragment was the primary site of hydration where specific solvent interactions were simulated through single- and triple- "water-phosphate" interactions, as water cluster's "tetrahedral dice" to a "trimeric motif" transformation as a partial de-clusterization was evident. Under all the hydration patterns considered in both static and dynamic descriptions of density functional theory, L-lipid/D-amino acid enantiomer adducts continued to be stable structures while in dynamic systems, water rearranged without getting "squeezed-out" in the process of recognition. In spite of the challenging computational realm of this multiscale problem, the ADMP simulated molecular interactions complying with polarized vibrational spectroscopy unraveled a novel route to chiral recognition and interfacial water structure.

Anomalous subsurface thermal behavior in tissue mimics upon near infrared irradiation mediated photothermal therapy.

Photothermal therapy using (Near Infrared) NIR region of EM spectrum is a fast emerging technology for cancer therapy. Different types of nanoparticles may be used for enhancing the treatment. Though the treatment protocols are developed based on experience driven estimated temperature increase in the tissue, it is not really known what spatiotemporal thermal behavior in the tissue is. In this work, this thermal behavior of tissue models is investigated with and without using nanoparticles. An increased temperature inside tissue compared to surface is observed which is counter intuitive from the present state of knowledge. It is shown from fiber level microstructure that this increased temperature leads to enhanced damage at the deeper parts of biomaterials. Nanoparticles can be utilized to control this temperature increase spatially. A multiple scattering based physical model is proposed to explain this counterintuitive temperature rise inside tissue. The results show promising future for better understanding and standardizing the protocols for photothermal therapy.