Membrane-water Interface Research Articles

Nitrated Fatty Acids Modulate the Physical Properties of Model Membranes and the Structure of Transmembrane Proteins.

Nitrated fatty acids (NO2 -FAs) act as anti-inflammatory signal mediators, albeit the molecular mechanisms behind NO2 -FAs' influence on diverse metabolic and signaling pathways in inflamed tissues are essentially elusive. Here, we combine fluorescence measurements with surface-specific sum frequency generation vibrational spectroscopy and coarse-grained computer simulations to demonstrate that NO2 -FAs alter lipid organization by accumulation at the membrane-water interface. As the function of membrane proteins strongly depends on both, protein structure as well as membrane properties, we consecutively follow the structural dynamics of an integral membrane protein in presence of NO2 -FAs. Based on our results, we suggest a molecular mechanism of the NO2 -FA in vivo activity: Driven by the NO2 -FA-induced lipid layer reorganization, the structure and function of membrane-associated (signaling) proteins is indirectly affected.

Solvent-Induced Molecular Folding and Self-Assembled Nanostructures of Tyrosine and Tryptophan Analogues in Aqueous Solution: Fascinating Morphology of High Order.

Hydrophobic derivatives of tyrosine and tryptophan, viz. octyl and dodecyl esters of tyrosine and octyl ester of tryptophan, are synthesized, and the interfacial and bulk properties in aqueous media are investigated as models for the membrane proteins. Molecular modeling by the density functional theory method is carried out to understand the molecular conformation and geometry for the purpose of determining the packing parameters. Water-induced molecular folding of the esters of both tyrosine and tryptophan, as observed using rotating frame nuclear Overhauser effect spectroscopy, indicates that the segregation of the hydrophobic and hydrophilic blocks in water is the key to the development of fascinating interfacial property displayed by the aromatic amino acid esters. The unusually high-order morphology of the aggregates, as observed using high-resolution transmission electron microscopy, is highly uncommon for single-chain amphiphiles and points to the fact that the self-assembly behavior of the present systems resembles that of block copolymers. The study of the growth of mesosized hollow aggregates with internal bilayer structures from tyrosine and tryptophan-based model systems would add to the understanding of biochemistry and biotechnology relevant to the cell membrane. The potential of biocompatible nanostructured motifs as the drug carriers is discussed. The highly functional role played by the aromatic amino acids at the membrane-water interface will be considered with the present amphiphilic models for future perspective.

Conformation change of an isotactic poly (N-isopropylacrylamide) membrane: Molecular dynamics.

In this work, isotactic Poly (N-Isopropylacrylamide)-PNIPAM-in neat water and in electrolyte solutions is studied by means of molecular dynamics simulations. This is done for an infinitely diluted oligomer and for an assembly of several PNIPAM chains arranged into a planar membrane configuration with a core-shell morphology. We employed two different force fields, AMBER (assisted model building with energy refinement) and OPLS-AA (all atom - optimized potentials for liquid simulations) in combination with extended simple point charge water. Despite the more water insoluble character of isotactic oligomers, our results support the existence of a coil to globule transition for the isolated 30-mer. This may imply the existence of an oligomer rich phase of coil-like structures in equilibrium with a water rich phase for temperatures close but below the coil to globule transition temperature, TΘ. However, the obtained coil structure is much more compact than that corresponding to the syndiotactic chain. Our estimations of TΘ are (308±5) K and (303±5) K for AMBER and OPLS-AA, respectively. The membrane configuration allows one to include chain-chain interactions, to follow density profiles of water, polymer, and solutes, and accessing the membrane-water interface tension. Results show gradual shrinking and swelling of the membrane by switching temperature above and below TΘ, as well as the increase and decrease of the membrane-water interface tension. Finally, concentration profiles for 1M NaCl and 1M NaI electrolytes are shown, depicting a strong salting-out effect for NaCl and a much lighter effect for NaI, in good qualitative agreement with experiments.

Sulpiride, Amisulpride, Thioridazine, and Olanzapine: Interaction with Model Membranes. Thermodynamic and Structural Aspects.

Neuroleptic drugs are widely applied in effective treatment of schizophrenia and related disorders. The lipophilic character of neuroleptics means that they tend to accumulate in the lipid membranes, impacting their functioning and processing. In this paper, the effect of four drugs, namely, thioridazine, olanzapine, sulpiride, and amisulpride, on neutral and negatively charged lipid bilayers was examined. The interaction of neuroleptics with lipids and the subsequent changes in the membrane physical properties was assessed using several complementary biophysical approaches (isothermal titration calorimetry, electron paramagnetic resonance spectroscopy, dynamic light scattering, and ζ potential measurements). We have determined the thermodynamic parameters, that is, the enthalpy of interaction and the binding constant, to describe the interactions of the investigated drugs with model membranes. Unlike thioridazine and olanzapine, which bind to both neutral and negatively charged membranes, amisulpride interacts with only the negatively charged one, while sulpiride does not bind to any of them. The mechanism of olanzapine and thioridazine insertion into the bilayer membrane cannot be described merely by a simple molecule partition between two different phases (the aqueous and the lipid phase). We have estimated the number of protons transferred in the course of drug binding to determine which of its forms, ionized or neutral, binds more strongly to the membrane. Finally, electron paramagnetic resonance results indicated that the drugs are localized near the water-membrane interface of the bilayer and presence of a negative charge promotes their burying deeper into the membrane.

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes.

Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques. The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes. These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains. Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.

Protonation of Long Chain Fatty Acids at the Membrane-Water Interface

Long-chain free fatty acids (FA) play an important role in several physiological and pathological processes such as lipid fusion, alteration of membrane permeability and fluidity, as well as regulation of enzyme and protein activity. FA-facilitated membrane proton transport (flip-flop) and FA-dependent proteinaceous proton transport (as in the case of mitochondrial uncoupling proteins) are governed by the difference between FA's pK and the pH in the immediate membrane vicinity1,2. So far, a quantitative understanding of the process is hampered by the shift in the pK value of carboxylic groups that occurs upon moving the FA from the aqueous solutions into the membrane. Its molecular origin is not understood, conceivably because the reported pKa values for the same FA are scattered between 5 and 10.5. Here we systematically obtained the dependence of apparent pKa values on chain length, number and position of double bonds by measuring the zeta-potential of liposomes reconstituted with FA at different pH. pKa ranged from 6.4 to 7.6. Combining the zeta potential measurements with molecular dynamic simulations, we found that pK is determined by the interplay between the energetic costs for burying a charged moiety into the bilayer and the hydrophobic interactions between the free and lipid-bound fatty acid chains.1. Pohl, E. E., Voltchenko, A. M. & Rupprecht, A. Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes. Biochim.Biophys.Acta 1778, 1292-1297 (2008).2. Rupprecht, A. et al. Role of the transmembrane potential in the membrane proton leak. Biophys.J. 98, 1503-1511 (2010).

Lipids, Neurotransmitters and their Receptors

Neurotransmission by chemical synapses is often described as a simple process, which starts from neurotransmitters release from presynaptic vesicles, is followed by neurotransmitters diffusion through the synaptic cleft to the opposite neuron, and is finally completed by the binding of neurotransmitters to their receptors, thus initializing the activation of a postsynaptic neuron. This description, however, may be too simplified given that there are numerous macromolecules in the synaptic cleft, which interact with neurotransmitters water-soluble domains in a non-specific way. This suggests that the binding of certain neurotransmitters to their receptor's binding site does not take place directly from the water phase but is instead membrane mediated. This possibility is largely overlooked in the literature. Our recent study of 13 neurotransmitters showed that neurotransmitters can be categorized into two groups: membrane-binding (e.g., dopamine, serotonin, epinephrine, norepinephrine, melatonin, and adenosine) and membrane-nonbinding molecules [1]. This categorization corresponds to the receptor types of their neurotransmitters: receptors of membrane-binding neurotransmitters are predominately G-protein coupled receptors with the entry to the binding pocket located close to the lipid membrane surface, while receptors of membrane-nonbinding neurotransmitters are ligand-gated ion channels with a binding pocket located far from the lipid membrane surface. These observations allowed us to postulate the existence of a membrane-mediated mechanism for neurotransmission where neurotransmitters first attach to the membrane surface and then through lateral diffusion find and enter their receptors binding site. Following this idea, we performed studies of dopamine and adenosine entry pathways to the binding pockets in their receptors. Using accelerated molecular dynamics simulations, free energy calculations, and atomistic molecular dynamics simulations we confirmed that the most probable path of dopamine and adenosine entry into their receptor takes place via the membrane-water interface. [1] P. Postila, I. Vattulainen, T. Rog. 2016. Selective effect of synaptic membrane on neurotransmission. Sci. Rep. 6:19345.

Disruption of gel phase lipid packing efficiency by sucralose studied with merocyanine 540

Sucralose, an artificial sweetener, displays very different behavior towards membranes than its synthetic precursor sucrose. The impact of both sugars on model dipalmitoylphosphatidylcholine model membranes was investigated using absorbance and flourescence spectroscopy and the membrane probe merocyanine 540. This probe molecule is highly sensitive to changes in membrane packing, microviscosity and polarity. This work focuses on the impact of sugars on the outer leaflet of unilamellar dipalmitoyl phosphatidylcholine model membranes. The choice of lipid permits access to the gel phase at room temperature and incorporation of the dye after liposome formation allows us to examine the direct impact of the sugar on the outer leaflet while maximizing the response of the dye to changes in the bilayer. The results demonstrate that sucrose has no impact on the packing efficiency of lipids in unilamellar DPPC vesicles in the gel phase. Conversely sucralose decreases the packing efficiency of lipids in the gel phase and results in decreased microviscosity and increased membrane fluidity, which may be as a result of water disruption at the membrane water interface.

Effect of piroxicam on lipid membranes: Drug encapsulation and gastric toxicity aspects

Uptake of piroxicam, a non-steroidal anti-inflammatory drug, from the intestines after oral intake is limited due to its low solubility and its wide use is associated with several side effects related to the gastrointestinal tract. In this study, all-atom molecular dynamics (MD) simulations and fluorescent spectroscopy were employed to investigate the interaction of piroxicam in neutral, zwitterionic, and cationic forms with lipid bilayers composed of phosphatidylcholine, cholesterol, and PEGylated lipids. Our study was aimed to assess the potential for encapsulation of piroxicam in liposomal carriers and to shed more light on the process of gastrointestinal tract injury by the drug. Through both the MD simulations and laser scanning confocal microscopy, we have demonstrated that all forms of piroxicam can associate with the lipid bilayers and locate close to the water-membrane interface. Conventional liposomes used in drug delivery are usually stabilized by the addition of cholesterol and have their bloodstream lifetime extended through the inclusion of PEGylated lipids in the formulation to create a protective polymer corona. For this reason, we tested the effect of these two modifications on the behavior of piroxicam in the membrane. When the bilayer was PEGylated, piroxicam localize to the PEG layer and within the lipid headgroup region. This suggests that PEGylated liposomes are capable of carrying a larger quantity of piroxicam than the conventional ones.

Independent adsorption of monovalent cations and cationic polymers at PE/PG lipid membranes

Synthetic cationic polymers constitute a wide class of polymeric biocides. Commonly their antimicrobial effect is associated to their interaction with bacterial membranes. In the present study we analyze the interaction of various cationic polymers with model bacterial membranes comprised of a mixture of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). We describe a polymer-membrane interaction as a process of modification of the surface charge. It is well known that small monovalent inorganic cations (Na+, K+) cannot overcharge the surface of a bilayer containing anionic lipids. In contrast, polycations are able to overcharge anionic membranes and demonstrate a very large input to the electric field distribution at the membrane-water interface. We aimed here to study the electrostatic effects associated with the interaction of polycations of different types with a model lipid membrane whose composition closely resembles that of bacterial membranes (PE:PG = 1:4). Four different cationic polymers (polyvinylamine, polyallylamine, poly-L-lysine and polyethylenimine) were adsorbed at a model PE/PG bilayer in MD simulations. Adsorption of sodium cations was inspected separately for PE/PG bilayers of different composition and cation’s binding parameters were determined. From computational experiments and consequent theoretical analysis we concluded that sodium adsorption at anionic binding sites does not depend on the presence of polycations. Therefore, we hypothesize that antimicrobial activity of the studied cationic polymers should depend on the ionic composition of the medium.

Evidence for Surface Recognition by a Cholesterol-Recognition Peptide

Two cholesterol recognition/interaction amino-acid consensus peptides, N-acetyl-LWYIKC-amide, and N-acetyl-CLWYIK-amide, have been coupled to exchangeable mimics of Chol (cholesterol) and Phos (1,2-dipalmitoyl-sn-glycerol-3-phospho-(1′rac-glycerol)) via disulfide bond formation. Equilibration between Chol and Phos via thiolate-disulfide interchange reactions has revealed that both peptides favor Chol as a nearest-neighbor in liquid-disordered (ld) bilayers to the same extent. In contrast, no Chol- or Phos-recognition could be detected by these peptides in analogous liquid-ordered (lo) bilayers. Fluorescence measurements of the tryptophan moiety have shown that both peptides favor the membrane-water interface. Taken together, these results provide strong evidence that the recognition behavior of the LWYIK motif is, fundamentally, a surface phenomenon but that partial penetration into the bilayer is also necessary.

Water Structure at the Lipid Multibilayer Surface: Anionic Versus Cationic Head Group Effects.

Membrane water interface is a potential reaction site for many biochemical reactions. Therefore, a molecular level understanding of water structure and dynamics that strongly depend on the chemical structure of lipid is prerequisite for elucidating the role of water in biological reactions on membrane surface. Recently, we carried out femtosecond infrared pump-probe studies of water structure and dynamics at multibilayer surfaces of zwitterionic phosphatidylcholine-analogue lipid ( J. Phys. Chem. Lett. 2016 , 7 , 741 ). Here, to further elucidate the anionic and cationic headgroup effects on water, we study vibrational dynamics of water on lipid multibilayers formed by anionic phospho-glycerol lipid molecules as well as by cationic choline-derivatized lipid molecules. We observed two significantly different vibrational lifetime components (very fast 0.5 ps and slow 1.9 ps) of the OD stretch mode of HOD molecules at the negatively charged phospho-lipid multibilayer whereas only one vibrational lifetime component (1.6 ps) was observed at the positively charged choline-derivatized lipid multibilayer. From the detailed analyses about the vibrational energy and rotational relaxations of HOD molecules in lipid multibilayers composed of anionic lipid with phosphate and cationic lipid without phosphate, the role of phosphate group in structuring water molecules at phospholipid membrane interface is revealed.

Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers

Lipid membrane surfaces can act as proton-collecting antennae, accelerating proton uptake by membrane-bound proton transporters. We investigated this phenomenon in lipid nanodiscs (NDs) at equilibrium on a local scale, analyzing fluorescence fluctuations of individual pH-sensitive fluorophores at the membrane surface by fluorescence correlation spectroscopy (FCS). The protonation rate of the fluorophores was ∼100-fold higher when located at 9- and 12-nm diameter NDs, compared to when in solution, indicating that the proton-collecting antenna effect is maximal already for a membrane area of ∼60 nm2. Fluorophore-labeled cytochrome c oxidase displayed a similar increase when reconstituted in 12 nm NDs, but not in 9 nm NDs, i.e., an acceleration of the protonation rate at the surface of cytochrome c oxidase is found when the lipid area surrounding the protein is larger than 80 nm2, but not when below 30 nm2. We also investigated the effect of external buffers on the fluorophore proton exchange rates at the ND membrane-water interfaces. With increasing buffer concentrations, the proton exchange rates were found to first decrease and then, at millimolar buffer concentrations, to increase. Monte Carlo simulations, based on a simple kinetic model of the proton exchange at the membrane-water interface, and using rate parameter values determined in our FCS experiments, could reconstruct both the observed membrane-size and the external buffer dependence. The FCS data in combination with the simulations indicate that the local proton diffusion coefficient along a membrane is ∼100 times slower than that observed over submillimeter distances by proton-pulse experiments (Ds ∼ 10−5cm2/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent.

Interaction of G protein coupled receptors and cholesterol

G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs.

Collagencin, an antibacterial peptide from fish collagen: Activity, structure and interaction dynamics with membrane

In this study, we first report characterization of collagencin, an antimicrobial peptide identified from fish collagen hydrolysate. The peptide completely inhibited the growth of Staphylococcus aureus at 1.88 mM. Although non-toxic up to 470 μM, collagencin was hemolytic at higher concentrations. The secondary structure of collagencin was mainly composed by β-sheet and β-turn as determined by CD measurements and molecular dynamics. The peptide is likely to form β-sheet structure under hydrophobic environments and interacts with both anionic (phosphatidylglycerol) and zwitterionic (phosphoethanolamine and phosphatidylcholine) lipids as shown with CD spectroscopy and molecular dynamics. The peptide formed several hydrogen bonds with both POPG and POPE lipids and remained at membrane–water interface, suggesting that collagencin antibacterial action follows a carpet mechanism. Collagenous fish wastes could be processed by enzymatic hydrolysis and transformed into products of high value having functional or biological properties. Marine collagens are a promising source of antimicrobial peptides with new implications in food safety and human health.

Molecular Insights into the Low Permeability Barrier of Gram-Negative Pathogens

Discovery of new antibiotics effective against Gram-negative bacteria is hampered by the low permeability barrier provided by two-membrane cell envelopes. The outer membrane (OM) is an asymmetric bilayer of lipopolysaccharides (LPS) and phospholipids and is highly impermeable to noxious compounds. Molecular characterization of the OM is essential to understand how antibiotics penetrate across the lipid barrier of Gram-negative bacteria as it can lead to new therapeutic strategies and rational design of novel compounds. We have used both all-atom and coarse-grained molecular dynamics simulations to characterize the fully assembled OM bilayer of Pseudomonas aeruginosa, a human pathogen resistant to most clinical antibiotics. In our model, we represent the most abundant LPS of P. aeruginosa corresponding to the A band O-antigen. Our results show that the LPS behaves differently from normal glycerophospholipids, affecting several physical properties of the LPS-containing bilayers. The extensive hydrogen bond network of the LPS decreases the lateral mobility of the lipid bilayer. In addition, the highly charged condensation closer to water-membrane interface, and the massive and extended carbohydrate head groups increase water permeation, which propagates to the aliphatic tails of the inner leaflet. This effect contributes to the low ordering of the lipids in the inner leaflet and leads to expanded area per lipid. Thermodynamic data suggest that the outer membrane by itself prevents the translocation of drugs. However, this barrier is dramatically decreased when assisted by porins and other transport channels in OM.

Folding and insertion thermodynamics of the transmembrane WALP peptide.

The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide's insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum-in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence.

The effect of cholesterol on the long-range network of interactions established among sea anemone Sticholysin II residues at the water-membrane interface.

Actinoporins are α-pore forming proteins with therapeutic potential, produced by sea anemones. Sticholysin II (StnII) from Stichodactyla helianthus is one of its most extensively characterized members. These proteins remain stably folded in water, but upon interaction with lipid bilayers, they oligomerize to form a pore. This event is triggered by the presence of sphingomyelin (SM), but cholesterol (Chol) facilitates pore formation. Membrane attachment and pore formation require changes involving long-distance rearrangements of residues located at the protein-membrane interface. The influence of Chol on membrane recognition, oligomerization, and/or pore formation is now studied using StnII variants, which are characterized in terms of their ability to interact with model membranes in the presence or absence of Chol. The results obtained frame Chol not only as an important partner for SM for functional membrane recognition but also as a molecule which significantly reduces the structural requirements for the mentioned conformational rearrangements to occur. However, given that the DOPC:SM:Chol vesicles employed display phase coexistence and have domain boundaries, the observed effects could be also due to the presence of these different phases on the membrane. In addition, it is also shown that the Arg51 guanidinium group is strictly required for membrane recognition, independently of the presence of Chol.

Water at Biological Phase Boundaries: Its Role in Interfacial Activation of Enzymes and Metabolic Pathways.

Many life-sustaining activities in living cells occur at the membrane-water interface. The pertinent questions that we need to ask are, what are the evolutionary reasons in biology for choosing the membrane-water interface as the site for performing and/or controlling crucial biological reactions, and what is the key physical principle that is very singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this chapter, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes through manipulation of the thermodynamic activity of water in the lipid-water interfacial region. The hypothesis is based on the fact that the surface pressure of a lipid monolayer is a direct measure of the thermodynamic activity of water at the lipid-water interface. Accordingly, the surface pressure-dependent activation or inactivation of interfacial enzymes is directly related to changes in the thermodynamic activity of interfacial water. Extension of this argument suggests that cells may manipulate conformations (and activities) of membrane-bound enzymes by manipulating the (re)activity of interfacial water at various locations in the membrane by localized compression or expansion of the interface. In this respect, cells may use the membrane-bound hormone receptors, lipid phase transition, and local variations in membrane lipid composition as effectors of local compression and/or expansion of membrane, and thereby local water activity. Several experimental data in the literature will be reexamined in the light of this hypothesis.

Experimental Demonstration of Localized Excess Protons at a WaterMembrane Interface

The widespread Mitchellian proton motive force equation has recently been revised with the proton-electrostatics localization hypothesis, which, for the first time, successfully elucidates the 30-year longstanding energetic conundrum of ATP synthesis in alkalophilic bacteria. To demonstrate the fundamental behavior of localized protons in a pure water-membrane-water system in relation to the newly derived pmf equation, excess protons and excess hydroxyl anions were generated by utilizing an i?½open-circuiti?½ water-electrolysis system and their distributions were tested using a proton-sensing aluminum membrane. The proton-sensing film placed at the membrane-water interface displayed dramatic localized proton activity while that placed into the bulk water phase showed no excess proton activity during the entire experiment. These observations clearly match with the prediction from the proton-electrostatics localization hypothesis that excess protons do not stay in water bulk phase; they localize at the water-membrane interface in a manner similar to the behavior of excess electrons in a conductor. This finding has significance not only in the science of bioenergetics but also in the fundamental understanding for the importance of water to life in serving as a proton conductor for energy transduction in living organisms.