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The cloud point temperatures of aqueous poly( N -isopropylacrylamide) (PNIPAM) and poly(ethylene) oxide (PEO) solutions were measured from pH 1.0 to pH 13.0 at a constant ionic strength of 100 mM. This ionic strength was reached by mixing the appropriate concentration of NaCl with either HCl or NaOH. The phase transition temperature of both polymers was nearly constant between pH 2.0 and 12.0. However, the introduction of 100 mM HCl (pH 1.0) led to an increase in the cloud point temperature, although this value was still lower than the cloud point temperature in the absence of salt. By contrast, the introduction of 100 mM NaOH (pH 13.0) caused a decrease in the cloud point temperature, both relative to adding 100 mM NaCl and adding no salt. Nuclear magnetic resonance (NMR) studies of these systems were performed below the cloud point temperature, and the chemical shifts closely tracked the corresponding changes in the phase transition temperature. Specifically, the introduction of 100 mM HCl caused the 1 H chemical shift to move downfield for the CH resonances from both PNIPAM and PEO, while 100 mM NaOH caused the same resonances to move upfield. Virtually no change in the chemical shift was seen between pH 2.0 and 12.0. These results are consistent with the idea that a sufficient concentration of H 3 O + led to polymer swelling compared to Na + , while substituting Cl - with OH - reduced swelling. Finally, classical all-atom molecular dynamics (MD) simulations were performed with a monomer and 5-mer corresponding to PNIPAM. The results correlated closely with the thermodynamic and spectroscopic data. The simulation showed that H 3 O + ions more readily accumulated around the amide oxygen moiety on PNIPAM compared with Na + . On the other hand, OH - was more excluded from the polymer surface than Cl - . Taken together, the thermodynamic, spectroscopic, and MD simulation data revealed that H 3 O + was less depleted from hydrophobic polymer/water interfaces than any of the monovalent Hofmeister metal cations or even Ca 2+ and Mg 2+ . As such, it should be placed on the far-right side of the cationic Hofmeister series. On the other hand, OH - was excluded from the interface and could be positioned in the anionic Hofmeister series between H 2 PO 4 - and SO 4 2- .

This study explores the interactions of the cis -[Ru(phen) 2 (Apy) 2 ] 2+ complex (RuApy, phen = 1,10-phenanthroline, Apy = 3,4-aminopyridine) with model lipid membranes to explain the role this complex plays in mitigating Aβ toxicity in PC12 neuronal cells. Fluorescence quenching, surface pressure isotherms in Langmuir monolayers, and infrared reflection-absorption analyses revealed that the positively charged RuApy interacts with the phosphate headgroups of monolayers, indirectly affecting ester carbonyl groups through hydrogen bonding with the amino group of the pyridine ligand of RuApy. These results offer a scenario for the protective effect of RuApy against Aβ toxicity in neuronal cells in which these interactions shield the electrostatic interactions of Aβ with lipid membranes, preserving membrane integrity and mitigating the deleterious influence of Aβ. This opens new avenues for antiamyloid strategies, focusing on compounds that prevent salt-bridge formation between bilayer membranes and amyloid proteins, aiding in the rational design of effective antiamyloid agents for therapeutic application.

Although post-translational lipidation is prevalent in eukaryotes, its impact on the liquid-liquid phase separation of disordered proteins is still poorly understood. Here, we examined the thermodynamic phase boundaries and kinetics of aqueous two-phase system (ATPS) formation for a library of elastin-like polypeptides modified with saturated fatty acids of different chain lengths. By systematically altering the physicochemical properties of the attached lipids, we were able to correlate the molecular properties of lipids to changes in the thermodynamic phase boundaries and the kinetic stability of droplets formed by these proteins. We discovered that increasing the chain length lowers the phase separation temperature in a sigmoidal manner due to alterations in the unfavorable interactions between protein and water and changes in the entropy of phase separation. Our kinetic studies unveiled remarkable sensitivity to lipid length, which we propose is due to the temperature-dependent interactions between lipids and the protein. Strikingly, we found that the addition of just a single methylene group is sufficient to allow tuning of these interactions as a function of temperature, with proteins modified with C7-C9 lipids exhibiting non-Arrhenius dependence in their phase separation, a behavior that is absent for both shorter and longer fatty acids. This work advances our theoretical understanding of protein-lipid interactions and opens avenues for the rational design of lipidated proteins in biomedical paradigms, where precise control over the phase separation is pivotal.

The effects of CaCl 2 and MgCl 2 on the cloud point temperature of two different elastin-like polypeptides (ELPs) were studied using a combination of cloud point measurements, molecular dynamics simulations, and infrared spectroscopy. Changes in the cloud point for the ELPs in aqueous divalent metal cation solutions were primarily governed by two competing interactions: the cation-amide oxygen electrostatic interaction and the hydration of the cation. In particular, Ca 2+ cations can more readily shed their hydration shells and directly contact two amide oxygens by the formation of ion bridges. By contrast, Mg 2+ cations were more strongly hydrated and preferred to partition toward the amide oxygens along with their hydration shells. In fact, although hydrophilic ELP V 5 A 2 G 3 was salted-out at low concentrations of MgCl 2 , it was salted-in at higher salt concentrations. By contrast, CaCl 2 salted the ELP sharply out of solution at higher salt concentrations because of the bridging effect.

Vibrational sum frequency spectroscopy (VSFS) and pressure-area Langmuir trough measurements were used to investigate the binding of alkali metal cations to eicosyl sulfate (ESO 4 ) surfactants in monolayers at the air/water interface. The number density of sulfate groups could be tuned by mixing the anionic surfactant with eicosanol. The equilibrium dissociation constant for K + to the fatty sulfate interface showed 10 times greater affinity than for Li + and approximately 3 times greater than for Na + . All three cations formed solvent shared ion pairs when the mole fraction of ESO 4 was 0.33 or lower. Above this threshold charge density, Li + formed contact ion pairs with the sulfate headgroups, presumably via bridging structures. By contrast, K + only bound to the sulfate moieties in solvent shared ion pairing configurations. The behavior for Na + was intermediate. These results demonstrate that there is not necessarily a correlation between contact ion pair formation and stronger binding affinity.

Weakly hydrated anions help to solubilize hydrophobic macromolecules in aqueous solutions, but small molecules comprising the same chemical constituents precipitate out when exposed to these ions. Here, this apparent contradiction is resolved by systematically investigating the interactions of NaSCN with polyethylene oxide oligomers and polymers of varying molecular weight. A combination of spectroscopic and computational results reveals that SCN - accumulates near the surface of polymers, but is excluded from monomers. This occurs because SCN - preferentially binds to the centre of macromolecular chains, where the local water hydrogen-bonding network is disrupted. These findings suggest a link between ion-specific effects and theories addressing how hydrophobic hydration is modulated by the size and shape of a hydrophobic entity., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Vibrational Stark shifts were explored in aqueous solutions of organic molecules with carbonyl- and nitrile-containing constituents. In many cases, the vibrational resonances from these moieties shifted toward lower frequency as salt was introduced into solution. This is in contrast to the blue-shift that would be expected based upon Onsager's reaction field theory. Salts containing well-hydrated cations like Mg 2+ or Li + led to the most pronounced Stark shift for the carbonyl group, while poorly hydrated cations like Cs + had the greatest impact on nitriles. Moreover, salts containing I - gave rise to larger Stark shifts than those containing Cl - . Molecular dynamics simulations indicated that cations and anions both accumulate around the probe in an ion- and probe-dependent manner. An electric field was generated by the ion pair, which pointed from the cation to the anion through the vibrational chromophore. This resulted from solvent-shared binding of the ions to the probes, consistent with their positions in the Hofmeister series. The "anti-Onsager" Stark shifts occur in both vibrational spectroscopy and fluorescence measurements.

Salt effects on the solubility of uncharged polymers in aqueous solutions are usually dominated by anions, while the role of the cation with which they are paired is often ignored. In this study, we examine the influence of three aqueous metal iodide salt solutions (LiI, NaI, and CsI) on the phase transition temperature of poly( N -isopropylacrylamide) (PNIPAM) by measuring the turbidity change of the solutions. Weakly hydrated anions, such as iodide, are known to interact with the polymer and thereby lead to salting-in behavior at low salt concentration followed by salting-out behavior at higher salt concentration. When varying the cation type, an unexpected salting-out trend is observed at higher salt concentrations, Cs + > Na + > Li + . Using molecular dynamics simulations, it is demonstrated that this originates from contact ion pair formation in the bulk solution, which introduces a competition for iodide ions between the polymer and cations. The weakly hydrated cation, Cs + , forms contact ion pairs with I - in the bulk solution, leading to depletion of CsI from the polymer-water interface. Microscopically, this is correlated with the repulsion of iodide ions from the amide moiety.

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.

Ion identity and concentration influence the solubility of macromolecules. To date, substantial effort has been focused on obtaining a molecular level understanding of specific effects for anions. By contrast, the role of cations has received significantly less attention and the underlying mechanisms by which cations interact with macromolecules remain more elusive. To address this issue, the solubility of poly( N -isopropylacrylamide), a thermoresponsive polymer with an amide moiety on its side chain, was studied in aqueous solutions with a series of nine different cation chloride salts as a function of salt concentration. Phase transition temperature measurements were correlated to molecular dynamics simulations. The results showed that although all cations were on average depleted from the macromolecule/water interface, more strongly hydrated cations were able to locally accumulate around the amide oxygen. These weakly favorable interactions helped to partially offset the salting-out effect. Moreover, the cations approached the interface together with chloride counterions in solvent-shared ion pairs. Because ion pairing was concentration-dependent, the mitigation of the dominant salting-out effect became greater as the salt concentration was increased. Weakly hydrated cations showed less propensity for ion pairing and weaker affinity for the amide oxygen. As such, there was substantially less mitigation of the net salting-out effect for these ions, even at high salt concentrations.

Herein, we show that Zn 2+ binds to phosphatidylserine (PS) lipids in supported lipid bilayers (SLBs), forming a PS-Zn 2+ complex with an equilibrium dissociation constant of ∼100 μM. Significantly, Zn 2+ binding to SLBs containing more than 10 mol % PS induces extensive reordering of the bilayer. This reordering is manifest through bright spots of high fluorescence intensity that can be observed when the bilayer contains a dye-labeled lipid. Measurements using atomic force microscopy (AFM) reveal that these spots represent three-dimensional unilamellar blebs. Bleb formation is ion specific, inducible by exposing the bilayer to μM concentrations of Zn 2+ but not Mg 2+ , Cu 2+ , Co 2+ , or Mn 2+ . Moreover, Ca 2+ can induce some blebbing at mM concentrations but not nearly as effectively as Zn 2+ . The interactions of divalent metal cations with PS lipids were further investigated by a combination of vibrational sum frequency spectroscopy (VSFS) and surface pressure-area isotherm measurements. VSFS revealed that Zn 2+ and Ca 2+ were bound to the phosphate and carboxylate moieties on PS via contact ion pairing, dehydrating the lipid headgroup, whereas Mg 2+ and Cu 2+ were bound without perturbing the hydration of these functional groups. Additionally, Zn 2+ was found to dramatically reduce the area per lipid in lipid monolayers, while Mg 2+ and Cu 2+ did not. Ca 2+ could also reduce the area per lipid but only when significantly higher surface pressures were applied. These measurements suggest that Zn 2+ caused lipid blebbing by decreasing the area per lipid on the side of the bilayer to which the salt was exposed. Such findings have implications for blebbing, fusion, oxidation, and related properties of PS-rich membranes in biological systems where Zn 2+ concentrations are asymmetrically distributed.

Phase separation of intrinsically disordered proteins (IDPs) is a remarkable feature of living cells to dynamically control intracellular partitioning. Despite the numerous new IDPs that have been identified, progress towards rational engineering in cells has been limited. To address this limitation, we systematically scanned the sequence space of native IDPs and designed artificial IDPs (A-IDPs) with different molecular weights and aromatic content, which exhibit variable condensate saturation concentrations and temperature cloud points in vitro and in cells. We created A-IDP puncta using these simple principles, which are capable of sequestering an enzyme and whose catalytic efficiency can be manipulated by the molecular weight of the A-IDP. These results provide a robust engineered platform for creating puncta with new, phase-separation-mediated control of biological function in living cells.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP 2 continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP 2 molecules in the top leaflet are fully mobile, while the PIP 2 molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP 2 species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP 2 flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H 2 O to D 2 O, it was shown that the primary interaction between PIP 2 and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP 2 . Moreover, PIP 2 immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP 2 on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.

Artificial water channels are synthetic molecules that aim to mimic the structural and functional features of biological water channels (aquaporins). Here we report on a cluster-forming organic nanoarchitecture, peptide-appended hybrid[4]arene (PAH[4]), as a new class of artificial water channels. Fluorescence experiments and simulations demonstrated that PAH[4]s can form, through lateral diffusion, clusters in lipid membranes that provide synergistic membrane-spanning paths for a rapid and selective water permeation through water-wire networks. Quantitative transport studies revealed that PAH[4]s can transport >10 9 water molecules per second per molecule, which is comparable to aquaporin water channels. The performance of these channels exceeds the upper bound limit of current desalination membranes by a factor of ~10 4 , as illustrated by the water/NaCl permeability-selectivity trade-off curve. PAH[4]'s unique properties of a high water/solute permselectivity via cooperative water-wire formation could usher in an alternative design paradigm for permeable membrane materials in separations, energy production and barrier applications.

The ability of cells or cell components to move in response to chemical signals is critical for the survival of living systems. This motion arises from harnessing free energy from enzymatic catalysis. Artificial model protocells derived from phospholipids and other amphiphiles have been made and their enzymatic-driven motion has been observed. However, control of directionality based on chemical cues (chemotaxis) has been difficult to achieve. Here we show both positive or negative chemotaxis of liposomal protocells. The protocells move autonomously by interacting with concentration gradients of either substrates or products in enzyme catalysis, or Hofmeister salts. We hypothesize that the propulsion mechanism is based on the interplay between enzyme-catalysis-induced positive chemotaxis and solute-phospholipid-based negative chemotaxis. Controlling the extent and direction of chemotaxis holds considerable potential for designing cell mimics and delivery vehicles that can reconfigure their motion in response to environmental conditions.

Aqueous two-phase system (ATPS) formation is the macroscopic completion of liquid-liquid phase separation (LLPS), a process by which aqueous solutions demix into 2 distinct phases. We report the temperature-dependent kinetics of ATPS formation for solutions containing a monoclonal antibody and polyethylene glycol. Measurements are made by capturing dark-field images of protein-rich droplet suspensions as a function of time along a linear temperature gradient. The rate constants for ATPS formation fall into 3 kinetically distinct categories that are directly visualized along the temperature gradient. In the metastable region, just below the phase separation temperature, T ph , ATPS formation is slow and has a large negative apparent activation energy. By contrast, ATPS formation proceeds more rapidly in the spinodal region, below the metastable temperature, T meta , and a small positive apparent activation energy is observed. These region-specific apparent activation energies suggest that ATPS formation involves 2 steps with opposite temperature dependencies. Droplet growth is the first step, which accelerates with decreasing temperature as the solution becomes increasingly supersaturated. The second step, however, involves droplet coalescence and is proportional to temperature. It becomes the rate-limiting step in the spinodal region. At even colder temperatures, below a gelation temperature, T gel , the proteins assemble into a kinetically trapped gel state that arrests ATPS formation. The kinetics of ATPS formation near T gel is associated with a remarkably fragile solid-like gel structure, which can form below either the metastable or the spinodal region of the phase diagram., Competing Interests: The authors declare no conflict of interest.

The anomalously high mobility of hydroxide and hydronium ions in aqueous solutions is related to proton transfer and structural diffusion. The role of counterions in these solutions, however, is often considered to be negligible. Herein, we explore the impact of alkali metal counter cations on hydroxide solvation and mobility. Impedance measurements demonstrate that hydroxide mobility is attenuated by lithium relative to sodium and potassium. These results are explained by ab initio molecular dynamics simulations and experimental vibrational hydration shell spectroscopy, which reveal substantially stronger ion pairing between OH - and Li + than with other cations. Hydration shell spectra and theoretical vibrational frequency calculations together imply that lithium and sodium cations have different effects on the delocalization of water protons donating a hydrogen bond to hydroxide. Specifically, lithium leads to enhanced proton delocalization compared with sodium. However, proton delocalization and the overall diffusion process are not necessarily correlated.

When a mixture of two salts in an aqueous solution contains a weakly and a strongly hydrated anion, their combined effect is nonadditive. Herein, we report such nonadditive effects on the lower critical solution temperature (LCST) of poly( N-isopropylacrylamide) (PNiPAM) for a fixed concentration of Na 2 SO 4 and an increasing concentration of NaI. Using molecular dynamics simulations and vibrational sum frequency spectroscopy, we demonstrate that at low concentrations of the weakly hydrated anion (I - ), the cations (Na + ) preferentially partition to the counterion cloud around the strongly hydrated anion (SO 4 2- ), leaving I - more hydrated. However, upon further increase in the NaI concentration, this weakly hydrated anion is forced out of solution to the polymer/water interface by sulfate. Thus, the LCST behavior of PNiPAM involves competing roles for ion hydration and polymer-iodide interactions. This concept can be generally applied to mixtures containing both a strongly and a weakly hydrated anion from the Hofmeister series.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that is thought to participate in the regulation of many physiological processes and may play a key role in several diseases. Herein, we found that Cu 2+ binds tightly to supported lipid bilayers (SLBs) containing S1P. Specifically, we demonstrated via fluorescence assays that Cu 2+ -S1P binding was bivalent and sensitive to the concentration of S1P in the SLB. In fact, the apparent equilibrium dissociation constant, K DApp , tightened by a factor of 132 from 4.5 μM to 34 nM as the S1P density was increased from 5.0 to 20 mol %. A major driving force for this apparent tightening was the more negative surface potential with increasing S1P concentration. This potential remained unaltered upon Cu 2+ binding at pH 7.4 because two protons were released for every Cu 2+ that bound. At pH 5.4, however, Cu 2+ could not outcompete protons for the amine and no binding occurred. Moreover, at pH 9.4, the amine was partially deprotonated before Cu 2+ binding and the surface potential became more positive on binding. The results for Cu 2+ -S1P binding were reminiscent of those for Cu 2+ -phosphatidylserine binding, where a carboxylate group helped to deprotonate the amine. In the case of S1P, however, the phosphate needed to bear two negative charges to facilitate amine deprotonation in the presence of Cu 2+ .

The interfacial water structure and phosphate group hydration of 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine monolayers were investigated at air/water interfaces. Both vibrational sum frequency spectroscopy (VSFS) and Langmuir monolayer compression measurements were made. The PC lipids oriented water molecules predominantly through their phosphate-choline (P-N) dipoles and carbonyl moieties. Upon the introduction of low concentrations of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a positively charged double chain surfactant, the TAP headgroups were attracted to the phosphate moieties on adjacent PC lipids. This attraction caused the monolayers to contract, expelling water molecules that were hydrogen bonded to the phosphate groups. Moreover, amplitude of the OH stretch signal decreased. At higher DOTAP concentrations, the positive charge on the monolayer caused an increase in the area per headgroup and water molecules in the near-surface bulk region became increasingly aligned. Under these latter conditions, the OH stretch amplitude was linearly proportional to the surface potential. By contrast, introducing 1,2-dioleoyl- sn-glycero-3-phosphatidylglycerol, a negatively charged lipid, did not change the area per lipid or the phosphate-water hydrogen bonding network. As the interfacial potential grew more negative, the OH stretch amplitude increased continuously. Significantly, changes in the interfacial water spectrum were independent of the chemistry employed to create the positive or negative interfacial potential. For example, Ca 2+ and tetracaine (both positively charged) disrupted the water structure similarly to low DOTAP concentrations, whereas SCN - and ibuprofen (both negatively charged) enhanced the water structure. These results suggest a direct correlation amongst the interfacial water structure, area per lipid, and surface charge density.

Pure aqueous electrolyte solutions display a minimum in surface tension at concentrations of 2 ± 1 mM. This effect has been a source of controversy since it was first reported by Jones and Ray in the 1930s. The Jones-Ray effect has frequently been dismissed as an artifact linked to the presence of surface-active impurities. Herein we systematically consider the effect of surface-active impurities by purposely adding nanomolar concentrations of surfactants to dilute electrolyte solutions. Trace amounts of surfactant are indeed found to decrease the surface tension and influence the surface chemistry. However, surfactants can be removed by repeated aspiration and stirring cycles, which eventually deplete the surfactant from solution, creating a pristine surface. Upon following this cleaning procedure, a reduction in the surface tension by millimolar concentrations of salt is still observed. Consequently, we demonstrate that the Jones-Ray effect is not caused by surface-active impurities.

Ibuprofen (IBU) interacts with phosphatidylcholine membranes in three distinct steps as a function of concentration. In a first step (<10 μM), IBU electrostatically adsorbs to the lipid headgroups and gradually decreases the interfacial potential. This first step helps to facilitate the second step (10-300 μM), in which hydrophobic insertion of the drug occurs. The second step disrupts the packing of the lipid acyl chains and expands the area per lipid. In a final step, above 300 μM IBU, the lipid membrane begins to solubilize, resulting in a detergent-like effect. The results described herein were obtained by a combination of fluorescence binding assays, vibrational sum frequency spectroscopy, and Langmuir monolayer compression experiments. By introducing trimethylammonium-propane, phosphatidylglycerol, and phosphatidylethanolamine lipids as well as cholesterol, we demonstrated that both the chemistry of the lipid headgroups and the packing of lipid acyl chains can substantially influence the interactions between IBU and the membranes. Moreover, different membrane chemistries can alter particular steps in the binding interaction.

The original version of this Article contained an error in the spelling of the author Woochul Song, which was incorrectly given as Woochul C. Song. This has been corrected in both the PDF and HTML versions of the Article.

Synthetic polymer membranes, critical to diverse energy-efficient separations, are subject to permeability-selectivity trade-offs that decrease their overall efficacy. These trade-offs are due to structural variations (e.g., broad pore size distributions) in both nonporous membranes used for Angstrom-scale separations and porous membranes used for nano to micron-scale separations. Biological membranes utilize well-defined Angstrom-scale pores to provide exceptional transport properties and can be used as inspiration to overcome this trade-off. Here, we present a comprehensive demonstration of such a bioinspired approach based on pillar[5]arene artificial water channels, resulting in artificial water channel-based block copolymer membranes. These membranes have a sharp selectivity profile with a molecular weight cutoff of ~ 500 Da, a size range challenging to achieve with current membranes, while achieving a large improvement in permeability (~65 L m -2  h -1  bar -1  compared with 4-7 L m -2  h -1  bar -1 ) over similarly rated commercial membranes.

On planet Earth, water is everywhere: the majority of the surface is covered with it; it is a key component of all life; its vapour and droplets fill the lower atmosphere; and even rocks contain it and undergo geomorphological changes because of it. A community of physical scientists largely drives studies of the chemistry of water and aqueous solutions, with expertise in biochemistry, spectroscopy and computer modelling. More recently, however, supramolecular chemists - with their expertise in macrocyclic synthesis and measuring supramolecular interactions - have renewed their interest in water-mediated non-covalent interactions. These two groups offer complementary expertise that, if harnessed, offer to accelerate our understanding of aqueous supramolecular chemistry and water writ large. This Review summarizes the state-of-the-art of the two fields, and highlights where there is latent chemical space for collaborative exploration by the two groups.

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Phosphatidylethanolamine (PE) is notoriously difficult to incorporate into model membrane systems, such as fluid supported lipid bilayers (SLBs), at high concentrations because of its intrinsic negative curvature. Using fluorescence-based techniques, we demonstrate that having fewer sites of unsaturation in the lipid tails leads to high-quality SLBs because these lipids help to minimize the curvature. Moreover, shorter saturated chains can help maintain the membranes in the fluid phase. Using these two guidelines, we find that up to 70 mol % PE can be incorporated into SLBs at room temperature and up to 90 mol % PE can be incorporated at 37 °C. Curiously, conditions under which three-dimensional tubules project outward from the planar surface as well as conditions under which domain formation occurs can be found. We have employed these model membrane systems to explore the ability of Ni 2+ to bind to PE. It was found that this transition metal ion binds 1000-fold tighter to PE than to phosphatidylcholine lipids. In the future, this platform could be exploited to monitor the binding of other transition metal ions or the binding of antimicrobial peptides. It could also be employed to explore the physical properties of PE-containing membranes, such as phase domain behavior and intermolecular hydrogen bonding.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-δ1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 ± 0.05 µM, similar to Kd,app values obtained by others. As previously observed, the PLC-δ1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein's capacity to interact with membranes.

Herein, we describe the development of oblique colloidal lithography (OCL) and establish a systematic patterning strategy for creating libraries of nanosized nonconcentric plasmonic structures. This strategy combines OCL, capillary force lithography, and several wet and ion etching steps. Hexagonal arrays of nonconcentric gold features were created on glass substrates with highly controllable geometric parameters. The size, geometry, and eccentricity of the gold features could be independently tuned by controlling the experimental conditions. Gaps within surface elements could be shrunk to as small as 30 nm, while the total patterned area was about l cm 2 . The goal was to devise a method that offers a high degree of control over the resolution and morphology of asymmetric structures without the need to resort to electron beam lithography. This technique also enabled the development of numerous surface patterns through the stepwise fabrication of separate elements. Complex features, including dots-surrounded nonconcentric targets, nonconcentric hexagram-disks, and nonconcentric annular aperture arrays, were demonstrated, and their optical properties were characterized. Indeed, spectroscopic studies and FDTD simulations demonstrated that Fano resonances could readily be generated by the nonconcentric gold features. Consequently, our patterning strategy should enable the high-throughput investigation of plasmonic coupling and Fano resonances as a function of the physical parameters of the elements within the nanopattern array.

The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca 2+ is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca 2+ is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca 2+ with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P 2 containing liposomes reveal that Ca 2+ as well as Mg 2+ reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P 2 lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC δ1-PH), is completely inhibited in the presence of Ca 2+ , while Mg 2+ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca 2+ binding to the PI(4,5)P 2 headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC δ1-PH and PI(4,5)P 2 , encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms.

Ions differ in their ability to salt out proteins from solution as expressed in the lyotropic or Hofmeister series of cations and anions. Since its first formulation in 1888, this series has been invoked in a plethora of effects, going beyond the original salting out/salting in idea to include enzyme activities and the crystallization of proteins, as well as to processes not involving proteins like ion exchange, the surface tension of electrolytes, or bubble coalescence. Although it has been clear that the Hofmeister series is intimately connected to ion hydration in homogeneous and heterogeneous environments and to ion pairing, its molecular origin has not been fully understood. This situation could have been summarized as follows: Many chemists used the Hofmeister series as a mantra to put a label on ion-specific behavior in various environments, rather than to reach a molecular level understanding and, consequently, an ability to predict a particular effect of a given salt ion on proteins in solutions. In this Feature Article we show that the cationic and anionic Hofmeister series can now be rationalized primarily in terms of specific interactions of salt ions with the backbone and charged side chain groups at the protein surface in solution. At the same time, we demonstrate the limitations of separating Hofmeister effects into independent cationic and anionic contributions due to the electroneutrality condition, as well as specific ion pairing, leading to interactions of ions of opposite polarity. Finally, we outline the route beyond Hofmeister chemistry in the direction of understanding specific roles of ions in various biological functionalities, where generic Hofmeister-type interactions can be complemented or even overruled by particular steric arrangements in various ion binding sites.

We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N -oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air-water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer-water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins.

A combination of Fourier transform infrared and phase transition measurements as well as molecular computer simulations, and thermodynamic modeling were performed to probe the mechanisms by which guanidinium (Gnd + ) salts influence the stability of the collapsed versus uncollapsed state of an elastin-like polypeptide (ELP), an uncharged thermoresponsive polymer. We found that the cation's action was highly dependent upon the counteranion with which it was paired. Specifically, Gnd + was depleted from the ELP/water interface and was found to stabilize the collapsed state of the macromolecule when paired with well-hydrated anions such as SO 4 2- . Stabilization in this case occurred via an excluded volume (or depletion) effect, whereby SO 4 2- was strongly partitioned away from the ELP/water interface. Intriguingly, at low salt concentrations, Gnd + was also found to stabilize the collapsed state of the ELP when paired with SCN - , which is a strong binder for the ELP. In this case, the anion and cation were both found to be enriched in the collapsed state of the polymer. The collapsed state was favored because the Gnd + cross-linked the polymer chains together. Moreover, the anion helped partition Gnd + to the polymer surface. At higher salt concentrations (>1.5 M), GndSCN switched to stabilizing the uncollapsed state because a sufficient amount of Gnd + and SCN - partitioned to the polymer surface to prevent cross-linking from occurring. Finally, in a third case, it was found that salts which interacted in an intermediate fashion with the polymer (e.g., GndCl) favored the uncollapsed conformation at all salt concentrations. These results provide a detailed, molecular-level, mechanistic picture of how Gnd + influences the stability of polypeptides in three distinct physical regimes by varying the anion. It also helps explain the circumstances under which guanidinium salts can act as powerful and versatile protein denaturants.

Phosphatidylserine (PS) has previously been found to bind Cu 2+ in a ratio of 1 Cu 2+ ion per 2 PS lipids to form a complex with an apparent dissociation constant that can be as low as picomolar. While the affinity of Cu 2+ for lipid membranes containing PS lipids has been well characterized, the structural details of the Cu-PS 2 complex have not yet been reported. Coordinating to one amine and one carboxylate moiety on two separate PS lipids, the Cu-PS 2 complex is unique among ion-lipid complexes in its ability to adopt both cis and trans conformations. Herein, we determine which stereoisomer of the Cu-PS 2 complex is favored in lipid bilayers using density functional theory calculations and electron paramagnetic resonance experiments. It was determined that a conformation in which the nitrogen centers are cis to each other is the preferred binding geometry. This is in contrast to the complex formed when two glycine molecules bind to Cu 2+ in bulk solution, where the cis and trans isomers exist in equilibrium, indicating that the lipid environment has a significant steric effect on the Cu 2+ binding conformation. These findings are relevant for understanding lipid oxidation caused by Cu 2+ binding to lipid membrane surfaces and will help us understand how ion binding to lipid membranes can affect their physical properties.

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

The interactions of two highly positively charged short peptide sequences with negatively charged lipid bilayers were explored by fluorescence binding assays and all-atom molecular dynamics simulations. The bilayers consisted of mixtures of phosphatidylglycerol (PG) and phosphatidylcholine (PC) lipids as well as a fluorescence probe that was sensitive to the interfacial potential. The first peptide contained nine arginine repeats (Arg9), and the second one had nine lysine repeats (Lys9). The experimentally determined apparent dissociation constants and Hill cooperativity coefficients demonstrated that the Arg9 peptides exhibited weakly anticooperative binding behavior at the bilayer interface at lower PG concentrations, but this anticooperative effect vanished once the bilayers contained at least 20 mol % PG. By contrast, Lys9 peptides showed strongly anticooperative binding behavior at all PG concentrations, and the dissociation constants with Lys9 were approximately 2 orders of magnitude higher than with Arg9. Moreover, only arginine-rich peptides could bind to the phospholipid bilayers containing just PC lipids. These results along with the corresponding molecular dynamics simulations suggested two important distinctions between the behavior of Arg9 and Lys9 that led to these striking differences in binding and cooperativity. First, the interactions of the guanidinium moieties on the Arg side chains with the phospholipid head groups were stronger than for the amino group. This helped facilitate stronger Arg9 binding at all PG concentrations that were tested. However, at PG concentrations of 20 mol % or greater, the Arg9 peptides came into sufficiently close proximity with each other so that favorable like-charge pairing between the guanidinium moieties could just offset the long-range electrostatic repulsions. This led to Arg9 aggregation at the bilayer surface. By contrast, Lys9 molecules experienced electrostatic repulsion from each other at all PG concentrations. These insights may help explain the propensity for cell penetrating peptides containing arginine to more effectively cross cell membranes in comparison with lysine-rich peptides.

Electrolytes interact with water in many ways: changing dipole orientation, inducing charge transfer, and distorting the hydrogen-bond network in the bulk and at interfaces. Numerous experiments and computations have detected short-range perturbations that extend up to three hydration shells around individual ions. We report a multiscale investigation of the bulk and surface of aqueous electrolyte solutions that extends from the atomic scale (using atomistic modeling) to nanoscopic length scales (using bulk and interfacial femtosecond second harmonic measurements) to the macroscopic scale (using surface tension experiments). Electrolytes induce orientational order at concentrations starting at 10 μM that causes nonspecific changes in the surface tension of dilute electrolyte solutions. Aside from ion-dipole interactions, collective hydrogen-bond interactions are crucial and explain the observed difference of a factor of 6 between light water and heavy water.

Herein, we demonstrate that Cu(2+) binds bivalently to phosphatidylethanolamine (PE), the second most abundant lipid in mammalian cells. The apparent equilibrium dissociation constant, K(DApp), for the Cu(2+)-PE complex at physiological pH is approximately 2 μM and is insensitive to the concentration of PE in the membrane. By contrast, at pH 10.0, where PE lipids bear a negative charge, K(DApp) decreases with increasing PE content and has a value of 150 nM for bilayers containing 70 mol % PE. The oxidation of double bonds in PE-containing bilayers can be monitored in the presence of Cu(2+). Strikingly, it was found that the oxidation rate is 8.2 times faster at pH 7.4 for bilayers containing 70 mol % PE than for pure phosphatidylcholine (PC) bilayers upon exposure of both to 70 μM Cu(2+) and 10 mM hydrogen peroxide. The rate of oxidation increases linearly with the PE content in the membrane. These results may help explain the high level of lipid oxidation in PE-containing membranes for neurodegenerative diseases and autism where the Cu(2+) concentration in the body is abnormally high.

Herein, we developed a new separation-based detection method that is capable of simultaneously identifying multiple competitively binding proteins for the same ligand on supported lipid bilayers (SLBs). This strategy used unlabeled target analyte proteins that bind to fluorescently tagged, lipid-conjugated ligands within the SLB. The protein-ligand binding complexes were then focused under an applied potential to different locations within the SLB based on each protein's size and charge. Both protein identity and relative surface concentration information could be obtained, simultaneously. Specifically, the competitive binding of streptavidin and goat anti-biotin for biotin-conjugated lipids was explored. It was found that streptavidin could inhibit the binding of goat anti-biotin antibodies for biotin-cap-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(biotin-cap-NBD-PE) lipids and that streptavidin more effectively outcompeted the anti-biotin antibody at lower protein concentrations. Also, modulating the chemical composition of the membrane helped control the ultimate focusing position and separation of the streptavidin-bound biotin, anti-biotin-bound biotin, and free biotin-conjugated lipid bands. The assay developed herein provides a simple and convenient strategy for simultaneously monitoring target analytes that bind to the identical ligand and may ultimately be useful in developing assays that help overcome problems associated with cross-reactivity.

Herein, the apparent equilibrium dissociation constant, K(Dapp), between Cu(2+) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), a negatively charged phospholipid, was measured as a function of PS concentrations in supported lipid bilayers (SLBs). The results indicated that K(Dapp) for Cu(2+) binding to PS-containing SLBs was enhanced by a factor of 17,000 from 110 nM to 6.4 pM as the PS density in the membrane was increased from 1.0 to 20 mol %. Although Cu(2+) bound bivalently to POPS at higher PS concentrations, this was not the dominant factor in increasing the binding affinity. Rather, the higher concentration of Cu(2+) within the double layer above the membrane was largely responsible for the tightening. Unlike the binding of other divalent metal ions such as Ca(2+) and Mg(2+) to PS, Cu(2+) binding does not alter the net negative charge on the membrane as the Cu(PS)2 complex forms. As such, the Cu(2+) concentration within the double layer region was greatly amplified relative to its concentration in bulk solution as the PS density was increased. This created a far larger enhancement to the apparent binding affinity than is observed by standard multivalent effects. These findings should help provide an understanding on the extent of Cu(2+)-PS binding in cell membranes, which may be relevant to biological processes such as amyloid-β peptide toxicity and lipid oxidation.

Herein, we use a combination of thermodynamic and spectroscopic measurements to investigate the interactions of Hofmeister anions with a thermoresponsive polymer, poly(N,N-diethylacrylamide) (PDEA). This amide-based polymer does not contain an NH moiety in its chemical structure and, thus, can serve as a model to test if anions bind to amides in the absence of an NH site. The lower critical solution temperature (LCST) of PDEA was measured as a function of the concentration for 11 sodium salts in aqueous solutions, and followed a direct Hofmeister series for the ability of anions to precipitate the polymer. More strongly hydrated anions (CO3(2-), SO4(2-), S2O3(2-), H2PO4(-), F(-), and Cl(-)) linearly decreased the LCST of the polymer with increasing the salt concentration. Weakly hydrated anions (SCN(-), ClO4(-), I(-), NO3(-), and Br(-)) increased the LCST at lower salt concentrations but salted the polymer out at higher salt concentrations. Proton nuclear magnetic resonance (NMR) was used to probe the mechanism of the salting-in effect and showed apparent binding between weakly hydrated anions (SCN(-) and I(-)) and the α protons of the polymer backbone. Additional experiments performed by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy found little change in the amide I band upon the addition of salt, which is consistent with very limited, if any, interactions between the salt ions and the carbonyl moiety of the amide. These results support a molecular mechanism for ion-specific effects on proteins and model amides that does not specifically require an NH group to interact with the anions for the salting-in effect to occur.

We demonstrate a procedure for the separation of enzymes based on their chemotactic response toward an imposed substrate concentration gradient. The separation is observed within a two-inlet, five-outlet microfluidic network, designed to allow mixtures of active (ones that catalyze substrate turnover) and inactive (ones that do not catalyze substrate turnover) enzymes, labeled with different fluorophores, to flow through one of the inlets. Substrate solution prepared in phosphate buffer was introduced through the other inlet of the device at the same flow rate. The steady-state concentration profiles of the enzymes were obtained at specific positions within the outlets of the microchannel using fluorescence microscopy. In the presence of a substrate concentration gradient, active enzyme molecules migrated preferentially toward the substrate channel. The excess migration of the active enzyme molecules was quantified in terms of an enrichment coefficient. Experiments were carried out with different pairs of enzymes. Coupling the physics of laminar flow of liquid and molecular diffusion, multiphysics simulations were carried out to estimate the extent of the chemotactic separation. Our results show that, with appropriate microfluidic arrangement, molecular chemotaxis leads to spontaneous separation of active enzyme molecules from their inactive counterparts of similar charge and size.

We have monitored the production of the negatively charged lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid acid (POPA), in supported lipid bilayers via the enzymatic hydrolysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC), a zwitterionic lipid. Experiments were performed with phospholipase D (PLD) in a Ca(2+) dependent fashion. The strategy for doing this involved using membrane-bound streptavidin as a biomarker for the charge on the membrane. The focusing position of streptavidin in electrophoretic-electroosmotic focusing (EEF) experiments was monitored via a fluorescent tag on this protein. The negative charge increased during these experiments due to the formation of POPA lipids. This caused the focusing position of streptavidin to migrate toward the negatively charged electrode. With the use of a calibration curve, the amount of POPA generated during this assay could be read out from the intact membrane, an objective that has been otherwise difficult to achieve because of the lack of unique chromophores on PA lipids. On the basis of these results, other enzymatic reactions involving the change in membrane charge could also be monitored in a similar way. This would include phosphorylation, dephosphorylation, lipid biosynthesis, and additional phospholipase reactions.

Salting out constants for triglycine were calculated for a series of Hofmeister salts using molecular dynamics simulations. Three variants of the peptide were considered with both termini capped, just the N -terminus capped, and without capping. The simulations were supported by NMR and FTIR measurements. The data provide strong evidence that earlier experimental values of salting out constants assigned to the fully capped peptide (as previously assumed) should have been assigned to the half-capped peptide instead. Therefore, these values cannot be used to directly establish Hofmeister ordering of ions at the peptide backbone, since they are strongly influenced by interactions of the ions with the negatively charged C -terminus.

Background: Detecting ligand-receptor binding on cell membrane surfaces is required to understand their function and behavior. Detection platforms can also provide an avenue for the development of medical devices and sensor biotechnology. The use of fluorescence techniques for such purposes is highly desirable as they provide high sensitivity. Herein, we describe a technique that utilizes the sensitivity of fluorescence without directly tagging the analyte of interest to monitor ligand-receptor interactions on supported lipid bilayers. The fluorescence signal is modulated according to the charge state of the target analyte. The binding event elicits protonation or deprotonation of pH-responsive reporter dyes embedded in the lipid bilayer., Methods: Supported lipid membranes containing ortho-conjugated rhodamine B-POPE (1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine), which fluoresces in its protonated but not in its deprotonated form, were utilized as sensor platforms for biotin-avidin and biotin-streptavidin binding events. The membranes contained 5 mol% biotin-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) as a capture ligand. Supported lipid bilayers were formed in the channels of microfluidic devices and the fluorescence intensity of the dye was monitored as protein was introduced., Results: The binding of avidin, which is positively charged at pH 7.2, made the bilayer surface charge more positive, which in turn deprotonated the ortho-rhodamine B dye, reducing its fluorescence. The binding of streptavidin, which is negatively charged at pH 7.2, had the opposite effect. Reducing the ionic strength of the analyte solution by removing 150 mM NaCl from the 10 mM phosphate buffered saline (PBS) solution raised the apparent pKa of the ortho-rhodamine B titration point by about 1 pH unit. This could be exploited in conjunction with bulk solution pH changes to turn the rhodamine B-POPE dye into a sensor for streptavidin involving a decrease, rather than an increase, in the fluorescence response, at pH values below streptavidin's pI value., Conclusions: This study demonstrates the ability to monitor ligand-receptor interactions on supported lipid bilayers through the protonation or deprotonation of reporter dyes for both negatively and positively charged analytes over a range of pH and ionic strength conditions. Specifically, the sensitivity and pH-operating range of this technique can be optimized by modulating the sensing conditions which are employed.