pH-induced Changes Research Articles

The pH-Induced Specific Area Changes of Unsaturated Lipids Deposited onto a Bubble Interface

In this work, we used an original experimental setup to examine the behavior of insoluble monolayers made with pH-sensitive lipids. Two kinds of unsaturated lipids were chosen: a cationic one (lipid 1) bearing an ammonium headgroup and an anionic one (lipid 2) terminated with an acidic phenol group. The lipids were deposited onto an air bubble interface maintained in an aqueous phase and, after stabilization, were subjected to a series of compressions performed at different pH values. These experiments disclosed a gradual increase in the specific area per molecule when lipids were neutralized. Imposing a pH variation at constant bubble volume also provided surface pressure profiles that confirmed this molecular behavior. As complementary characterization, dilatational rheology disclosed a phase transition from a purely elastic monophasic system to a viscoelastic two-phase system. We hypothesized that this unexpected increase in the specific area with lipid neutralization is related to the presence of unsaturations in each of the two branches of the hydrophobic tails that induce disorder, thereby increasing the molecular area at the interface. Application of the two-dimensional Volmer equation of state allowed the generation of quantitative values for the specific areas that showed variations with pH. It also allowed the determination of apparent pKa values, which are affected by both the electrostatic potential within the monolayer and the affinity of the lipid polar head for the aqueous phase.

PH에 따른 투명 및 써클 소프트콘택트렌즈의 산소투과율 변화

pH에 따른 투명 및 써클 소프트콘택트렌즈의 산소투과율 변화

Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering.

Messenger ribonucleic acid (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clinical practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clinical efficacy. In this study, pH-induced changes in internal molecular organization and overall physicochemical characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were determined for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate determination of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ determination of the structural response of the lipoplex systems to changes of the environmental pH similar to that observed for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.

Study two kind different catalytic behaviors for K4H1.2[Co0.6(H2O)0.6SiW11.4O39.4]-cocatalyzed visible light driven water oxidation in pH 1–7 media

Efficient water oxidation remains a particular challenge in water splitting that calls for developing robust and economic water oxidation catalysts (WOCs), preferably based on earth-abundant transition metals. Herein, we report a robust and noble metal-free catalytical system using BiVO4 as an effective light-harvesting material, a cobalt-containing polyoxometalate K4H1.2[Co0.6(H2O)0.6SiW11.4O39.4] (1) as molecular cocatalyst, and NaIO3 as a sacrificial electron acceptor for visible-light-driven water oxidation in a wide pH range 1–7. The combination of 1 + BiVO4 samples gives better oxygen evolution activities than that of pristine BiVO4. A turnover number (TON) of 326.3, apparent quantum efficiency (AQE) of 10.0%, and an O2 yield of 21.8% are obtained under pH 4. This work investigates two kind different catalytic behaviors for 1-cocatalyzed visible light driven water oxidation at pH 1–7 media, which exhibits a pH-induced dynamic change on the catalytic active species. Catalytic behavior, XPS, SEM and TEM collectively testify that 1 remains intact during photocatalytic water oxidation under pH 4–5. Conversely, under pH 1–3 and 6–7, the heterogeneous CoOx originated from 1 is formed on the BiVO4 surface, which is responsible for the enhancement of BiVO4 photocatalytic activity. Our work uncovers the borderline between homogeneous and heterogeneous WOCs using a cobalt-based polyoxometalate at different pH media, which helps to the study of stability for polyoxometalates when semiconducting material is used as the light-harvesting material.

Fluctuating seawater pCO2/pH induces opposing interactions with copper toxicity for two intertidal invertebrates

Global ocean pCO2 is increasing as a result of anthropogenic CO2 emissions, driving a decline in seawater pH. However, coastal waters already undergo fluctuations in pCO2/pH conditions over far shorter timescales, with values regularly exceeding those predicted for the open ocean by the year 2100. The speciation of copper, and therefore its potential toxicity, is affected by changing seawater pH, yet little is known concerning how present-day natural fluctuations in seawater pH affect copper toxicity to marine biota. Here, we test the hypothesis that a fluctuating seawater pCO2/pH regime will alter the responses of the mussel Mytilus edulis and the ragworm Alitta virens to sub-lethal copper, compared to a static seawater pCO2/pH scenario. Mussels and worms were exposed to 0.1 and 0.25 μM copper respectively, concentrations determined to produce comparable toxicity responses in these species, for two weeks under a fluctuating 12-hour pCO2/pH cycle (pH 8.14–7.53, pCO2 445–1747 μatm) or a static pH 8.14 (pCO2 432 μatm) treatment. Mussels underwent a haemolymph acidosis of 0.1–0.2 pH units in the fluctuating treatments, alongside two-fold increases in the superoxide dismutase activity and DNA damage induced by copper, compared to those induced by copper under static pH conditions. Conversely, ragworms experienced an alkalosis of 0.3 pH units under fluctuating pH/pCO2, driven by a two-fold increase in coelomic fluid bicarbonate. This mitigated the copper-induced oxidative stress to slightly reduce both antioxidant activity and DNA damage, relative to the static pH + copper treatment. These opposing responses suggest that differences in species acid-base physiology were more important in determining toxicity responses than the pH-induced speciation change. With variability in seawater chemistry predicted to increase as climate change progresses, understanding how fluctuating conditions interact with the toxicity of pH-sensitive contaminants will become more crucial in predicting their risk to coastal biota.

β-Lactoglobulin Adsorption Layers at the Water/Air Surface: 4. Impact on the Stability of Foam Films and Foams

The complexity and high sensitivity of proteins to environmental factors give rise to a multitude of variables, which affect the stabilization mechanisms in protein foams. Interfacial and foaming properties of proteins have been widely studied, but the reported unique effect of pH, which can be of great interest to applications, has been investigated to a lesser extent. In this paper, we focus on the impact of pH on the stability of black foam films and corresponding foams obtained from solutions of a model globular protein—the whey β-lactoglobulin (BLG). Foam stability was analyzed utilizing three characteristic parameters (deviation time, transition time and half-lifetime) for monitoring the foam decay, while foam film stability was measured in terms of the critical disjoining pressure of film rupture. We attempt to explain correlations between the macroscopic properties of a foam system and those of its major building blocks (foam films and interfaces), and thus, to identify structure-property relationships in foam. Good correlations were found between the stabilities of black foam films and foams, while relations to the properties of adsorption layers appeared to be intricate. That is because pH-dependent interfacial properties of proteins usually exhibit an extremum around the isoelectric point (pI), but the stability of BLG foam films increases with increasing pH (3–7), which is well reflected in the foam stability. We discuss the possible reasons behind these intriguingly different behaviors on the basis of pH-induced changes in the molecular properties of BLG, which seem to be determining the mechanism of film rupture at the critical disjoining pressure.

Nitric Oxide Synthase Oxygenase in Thin Layer-By-Layer Polymeric Films: Does the pH at Which the Film Is Built Affect the Direct Electron Transfer or the Related NOS-Mediated Catalysis?

We prepared thin films containing nitric oxide synthase (NOS) oxygenase enzymes and polyethyleneimine (PEI) using the layer-by-layer film building strategy. The charge-based layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films. During the film building process, we used NOS oxygenase enzyme from solutions at two distinct pHs (pH=7.0 and pH=8.6) to study the effect of pH on enzyme loading in the thin polymeric film. We studied direct electron transfer to NOS oxygenase in the two kinds of films at physiologic pH. We show that NOS oxygenase enzymes maintain their overall integral structure in PEI films in both pH conditions, and exhibit relatively fast but similar electron transfer rates. Using thin film coulometry, we show that the enzyme loading from pH 8.6 is almost 4-times more than the amount loaded in films prepared using NOS oxygenase solutions at pH=7.0.To closely examine the effect of pH during the layer-by-layer film building on the trapped NOS oxygenase enzymes, we studied the catalytic reduction of exogenous nitric oxide analyte mediated by NOS oxygenases in PEI films. We used this property to compare and contrast the catalytic activity of the films of NOS enzymes prepared at pH 7.0 and those prepared at pH 8.6. Our results show that there is a distinct increase in catalytic activity of enzymes adsorbed from pH 8.6 compared to those prepared at pH=7.0. We also show that the measured differential loading amnounts in the two kinds of films cannot explain the observed increase in mediated catalytic activity. In this regard, we show that NOS oxygenase films constructed at pH 8.6 exhibit significantly higher catalytic efficiency (kcat /KM) compared to films constructed at pH 7.0. To better understand the change in catalytic activity, we used detailed Fourier Transform Infrared (FTIR) spectroscopic analysis of the thin films to closely examine the structural integrity of the trapped NOS oxygenases in the two sets of films. Deconvolution methods using FTIR data suggest that although NOS oxygenase enzymes maintain their overall structure, we observe distinct pH-dependent conformational changes, which includes a decrease in α-helix contributions in the NOS protein adsorbed at pH 8.6. The observed changes in the secondary structure apparently add to the disordering of protective loops that affect a hydrophobic pocket trap that is critical for NO binding during catalysis.This finding is in line with the observed change in NO affinity for the trapped NOS enzymes as evidenced by the distinct change in catalytic efficiency of NOS oxygenases in the two sets of films. This work shows that while the electron transfer to NOS enzymes in PEI films is not affected by pH in which the layer-by-layer film fabrication occurs, the subtle pH-induced backbone changes provides a structural basis for the higher specific catalytic electrochemical reduction of NO by NOS oxygenases in PEI films constructed at pH 8.6.

PH-Induced Changes in Polypeptide Conformation: Force-Field Comparison with Experimental Validation.

Microsecond-long all-atom molecular dynamics (MD) simulations, circular dichroism, laser Doppler velocimetry, and dynamic light-scattering techniques have been used to investigate pH-induced changes in the secondary structure, charge, and conformation of poly l-lysine (PLL) and poly l-glutamic acid (PGA). The employed combination of the experimental methods reveals for both PLL and PGA a narrow pH range at which they are charged enough to form stable colloidal suspensions, maintaining their α-helix content above 60%; an elevated charge state of the peptides required for colloidal stability promotes the peptide solvation as a random coil. To obtain a more microscopic view on the conformations and to verify the modeling performance, peptide secondary structure and conformations rising in MD simulations are also examined using three different force fields, i.e., OPLS-AA, CHARMM27, and AMBER99SB*-ILDNP. Ramachandran plots reveal that in the examined setup the α-helix content is systematically overestimated in CHARMM27, while OPLS-AA overestimates the β-sheet fraction at lower ionization degrees. At high ionization degrees, the OPLS-AA force-field-predicted secondary structure fractions match the experimentally measured distribution most closely. However, the pH-induced changes in PLL and PGA secondary structure are reasonably captured only by the AMBER99SB*-ILDNP force field, with the exception of the fully charged PGA in which the α-helix content is overestimated. The comparison to simulations results shows that the examined force fields involve significant deviations in their predictions for charged homopolypeptides. The detailed mapping of secondary structure dependency on pH for the polypeptides, especially finding the stable colloidal α-helical regime for both examined peptides, has significant potential for practical applications of the charged homopolypeptides. The findings raise attention especially to the pH fine tuning as an underappreciated control factor in surface modification and self-assembly.

PH-induced changes in fungal abundance and composition affects soil heterotrophic nitrification after 30 days of artificial pH manipulation

Heterotrophic nitrification occurs extensively and is important for NO3– production in acidic soils. However, whether low soil pH stimulates heterotrophic nitrification is unknown and the potential microbial driver is unclear. Thus, a pH gradient (3.5, 4.5, 5.5, 6.5, 7.5) was manipulated in forest (SF with initial pH 4.5) and cropland (SC with initial pH 5.5) soils in subtropical China to illustrate the effect of soil pH on heterotrophic nitrification. After 30 days of pH regulation, 1% C2H2 was used to inhibit autotrophic nitrification and reveal heterotrophic nitrification via 15N-labelling experiments. During 30 days of pH regulation, soil microbial properties (e.g. gene abundance and composition of fungi and bacteria) were also determined to study the potential microbial driver of heterotrophic nitrification. The results showed that the gross heterotrophic nitrification rates increased from <0.3 mg N kg−1 day−1 in the pH 7.5 treatments to >1 mg N kg−1 day−1 in the pH 3.5 treatments, and the contribution of heterotrophic nitrification to the total nitrification was enhanced to more than 60% in the low pH treatments in both SF and SC. With soil acidification, more organic than inorganic N substrate was used in heterotrophic nitrification. Fungi showed a positive correlation with the gross heterotrophic nitrification rate (P < 0.01) and with the contribution of heterotrophic 15N-NO3– production to total 15N-NO3– production (P < 0.01), suggesting that fungi were the dominant heterotrophic nitrifiers in acidic soils. In addition, Phialocephala, Chloridium, and Tararomyces may have the potential for heterotrophic nitrification in our studied acidic soils. The present study suggested the decreasing soil pH could affect fungal abundance and composition, in turn, stimulate heterotrophic nitrification after a short term of pH regulation.

Influence of pH on the stability and structure of single casein microparticles

The aim of the study is to investigate the influence of pH on the structure and stability of casein microparticles prepared by depletion flocculation of casein micelles in solution followed by drying into a solid pellet. We have developed a microfluidic sample environment to perform microscopic single particle analyses. The setup allows a detailed investigation of the particle size and the internal structure after changes of milieu conditions. While the particles remain stable in HCl and ultrapure water, the addition of NaOH leads to a two-step swelling process, which finally results in degradation of the microparticle at pH values ≥ 11. At moderate pH values, the structural changes take several hours at pH 8, a few minutes at pH 11 and a few seconds at pH 14. The pH-induced size changes of the microparticle are described with a dynamic model assuming two independent solvent inflows occurring at different times and depending on a constant rate coefficient and the current particle volume. The model analysis shows that from pH 8 to pH 14 the characteristic times of the swelling processes decrease by three orders of magnitude while the rate coefficients increase by four orders of magnitude. Microparticles consist of building blocks, which are microscopically detectable as a microstructure with a characteristic size of a few μm. These building blocks are stretched in the first phase of the swelling process. This leads to the assumption that at basic pH values the microstructure swells in a first step and then in a second step the swollen microparticle disintegrates due to the weakening of the stabilizing contacts between the building blocks.

Laboratory investigation on the effects of pH-induced changes on geotechnical characteristics of clay soil

ABSTRACT This paper attempts to study the effects of acidic and alkaline waters on the geotechnical properties of a particular type of clay with low plasticity from the northeast areas of Isfahan that have been evaluated with pH values adjusted from 3 to 8. The remoulded specimens were placed in moulds under the influence of acid and alkaline rains with different pH values and years of precipitation. Atterberg limits tests, soil permeability coefficient, California bearing ratio (CBR) test and unconfined compressive strength (UCS) of soil specimens with various pH values were used to evaluate soil mechanical changes relative to soil with neutral acidity. In order to identify internal structural mechanisms of soil before and after artificial raining on the specimens, scanning electron microscopy and pH drainage tests of specimens were performed. The results of the interaction of rain and soil with shifting from neutral values were evaluated over a period of precipitation fluxes from 1 to 20 years. In overall, the more is the acidity or alkalinity of the artificial rain, the more will increase the Atterberg limits and the soil permeability compared to the soil specimens under artificial rain with pH = 7; and the decreasing trend is observed in UCS values and CBR.

Lipophilicity matters – A new look at experimental plant uptake data from literature

Peer-reviewed Transpiration Stream Concentration Factor (TSCF) values were analysed to elucidate whether pH-induced changes in lipophilicity can explain some of the variability in reported TSCF and whether a potential relationship between lipophilicity and TSCF can be described by a simple mathematical model. The data set for this investigation combined TSCF values of 42 non-ionisable and ionisable compounds from hydroponic tests with intact plants and publicly available lipophilicity data for the tested compounds. The data set was not homogenous in terms of molecular weight of the tested compounds, plant species used for testing and experimental conditions, but a strong effect of one of these factors on variation in reported TSCF was not detected. Variation in TSCF was high for the same or similar predicted octanol/water partitioning coefficient (log P) but could be reduced by considering octanol/water distribution coefficients (log D) instead. The TSCF data set was split into a training and a test data set in order to identify and test a best-fit model describing the relationship between log D and TSCF. Comparing different types of models (linear, sigmoidal, Gaussian), the Gaussian model fitted to the training data set after removal of two outliers was identified as best-fit model based on visual assessment and fit statistics (RMSE = 0.20, NSE = 0.57, R = 0.75 (p < 0.001)). The 95% confidence interval around the best-fit model contained about 70% of data points in the training set and the test set, respectively. In conclusion, compound lipophilicity expressed as log D is a more appropriate descriptor of uptake by plant roots and subsequent translocation than log P when ionisable compounds are considered. Furthermore, findings in this study suggest that a relationship exists between log D and TSCF for uptake tests with intact plants which can be described by a simple bell-shaped Gaussian model.

PH Responsiveness of Hexosomes and Cubosomes for Combined Delivery of Brucea javanica Oil and Doxorubicin.

We report pH-responsive liquid crystalline lipid nanoparticles, which are dual-loaded by Brucea javanica oil (BJO) and doxorubicin hydrochloride (DOX) and display a pH-induced inverted hexagonal (pH = 7.4) to cubic (pH = 6.8) to emulsified microemulsion (pH = 5.3) phase transition with a therapeutic application in cancer inhibition. BJO is a traditional herbal medicine that strongly inhibits the proliferation and metastasis of various cancers. Doxorubicin is an antitumor drug, which prevents DNA replication and hampers protein synthesis through intercalation between the base pairs of the DNA helices. Its dose-dependent cardiotoxicity imposes the need for safe delivery carriers. Here, pH-induced changes in the structural and interfacial properties of designed multicomponent drug delivery (monoolein-oleic acid-BJO-DOX) systems are determined by synchrotron small-angle X-ray scattering and the Langmuir film balance technique. The nanocarrier assemblies display good physical stability in the studied pH range and adequate particle sizes and ζ-potentials. Their interaction with model lipid membrane interfaces is enhanced under acidic pH conditions, which mimic the microenvironment around tumor cells. In vitro cytotoxicity and apoptosis studies with BJO-DOX dual-loaded pH-switchable liquid crystalline nanoparticles are performed on the human breast cancer Michigan Cancer Foundation-7 (MCF-7) cell line and MCF-7 cells with doxorubicin resistance (MCF-7/DOX), respectively. The obtained pH-sensitive nanomedicines exhibit enhanced antitumor efficacy. The performed preliminary studies suggest a potential reversal of the resistance of the MCF-7/DOX cells to DOX. These results highlight the necessity for further understanding the link between the established pH-dependent drug release profiles of the nanocarriers and the role of their pH-switchable inverted hexagonal, bicontinuous cubic, and emulsified microemulsion inner organizations for therapeutic outcomes.

PH titration of β-lactoglobulin monitored by laser-based Mid-IR transmission spectroscopy coupled to chemometric analysis.

A novel external cavity-quantum cascade laser (EC-QCL)-based setup for mid-IR transmission spectroscopy in the amide I and amide II region was employed for monitoring pH-induced changes of protein secondary structure. pH titration of β-lactoglobulin revealed unfolding of the native β-sheet secondary structure occurring at basic pH. Chemometric analysis of the dynamic IR spectra was performed by multivariate curve resolution-alternating least squares (MCR-ALS). Using this approach, spectral and abundance distribution profiles of the conformational transition were obtained. A proper post-processing procedure was implemented allowing to extract information about pure protein spectra and spurious signals that may interfere in the interpretation of the system. This work demonstrates the potential and versatility of the EC-QCL-based IR transmission setup for flow-through applications, benefitting from the high available optical path length.

CpHMD-Then-QM/MM Identification of the Amino Acids Responsible for the Anabaena Sensory Rhodopsin pH-Dependent Electronic Absorption Spectrum.

Anabaena Sensory Rhodopsin (ASR), a microbial photoactive protein featuring the retinal chromophore in two different conformations, exhibits a pH-dependent electronic absorption spectrum. Using the recently developed CpHMD-then-QM/MM multiscale protocol applied to ASR embedded in a membrane model, the pH-induced changes in its maximum absorption wavelength have been reproduced and analyzed. While the acidic tiny red-shift is essentially correlated with the deprotonation of an aspartic acid located on the ASR extracellular side, the larger blue-shift experimentally reported at pH values larger than 5 involves a cluster of titrating residues sitting on the cytoplasmic side. The ASR pH-dependent spectrum is the consequence of the competitive stabilization of retinal ground and excited states by the protein electrostatic potential.

An insight into pH-induced changes in FAD conformational structure by means of time-resolved fluorescence and circular dichroism.

Optical properties of flavin adenine dinucleotide (FAD) moiety are widely used nowadays for biotechnological applications. Given the fundamental role played by FAD, additional structural information about this enzymatic cofactor can be extremely useful in order to obtain a greater insight into its functional role in proteins. For this purpose, we have investigated FAD behaviour in aqueous solutions at different pH values by a novel approach based on the combined use of time-resolved fluorescence and circular dichroism spectroscopies. The results showed that pH strongly affects time-resolved fluorescence emission and the analysis allowed us to detect a three-component decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. Circular dichroism data were analyzed by a multi-Gaussian fitting procedure and the trends of properly chosen parameters confirmed pH-depending changes. The comparison between the results obtained by these two optical techniques allowed us to improve the significance of the outcome of circular dichroism. This combined approach may provide a useful tool for biotechnological investigation.

PH-induced morphological changes of proteinaceous viral shells

Changes in environmental pH can induce morphological changes in empty proteinaceous shells of bacteriophages in vitro that are very similar to changes occurring in viral capsids in vivo after encapsidation of DNA. These changes in capsid shape and size cannot be explained with a simple elastic model alone. We propose a new theoretical framework that combines the elasticity of thin icosahedral shells with the pH dependence of capsid charge distribution. Minimization of the sum of elastic and electrostatic free energies leads to equilibrium shapes of viral shells that depend on a single elastic parameter and the detailed configuration of the imbedded protein charges. Based on the in vitro shell reconstructions of bacteriophage HK97 we elucidate the details of how the reversible transition between Prohead II and Expansion Intermediate II states of the HK97 procapsid is induced by pH changes, as well as some other features of the bacteriophage maturation.

Protection of anthocyanin-rich extract from pH-induced color changes using water-in-oil-in-water emulsions

The food, cosmetics, and personal care industries are increasingly interested in replacing synthetic colorants with natural alternatives. Anthocyanin is a renewable and sustainable source of plant-derived pigment. However, their application in commercial products is often limited because of their susceptibility to chemical degradation, which leads to color fading and/or a change in hue. In this study, we examined the possibility of protecting anthocyanin from degradation by encapsulating them within the inner water phase of a water-in-oil-in-water (W/O/W) emulsion. Polyglycerol polyricinoleate (PGPR) was used as a hydrophobic emulsifier, while quillaja saponin was used as a hydrophilic emulsifier. First, W/O emulsions containing 20% aqueous phase and 80% oil phase (PGPR in corn oil) were prepared using a microfluidizer. Second, W/O/W emulsions were prepared by blending 20% W/O emulsion with 80% aqueous phase (saponin in water) using a high-shear mixer. Changes in color, particle size, charge, and anthocyanin leakage of the emulsions were measured when the external aqueous phase was changed from pH 7 to 3. Anthocyanin encapsulation significantly slowed down pH-induced color changes, possibly because the internal aqueous phase had a different pH to the external one. Anthocyanin transfer between aqueous phases depended on pH, temperature, and initial location. Our results suggest that multiple emulsions may be useful for encapsulation and protection of natural colors.

Investigation of a KcsA Cytoplasmic pH Gate in Lipoprotein Nanodiscs.

The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane-cytoplasm interface (TM2 gate), and in the cytoplasmic C-terminal domain (CTD), an amphiphilic four-helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH-induced open-to-closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl-protonated at Leu/Val residues to allow observation of both states by NMR, and spin-labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0-5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin-spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated "closed" channels. We extended these findings for isolated CTD-derived peptides to full-length KcsA and have established a contribution of the CTD to KcsA pH-controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.

Structural Basis for the Lipid-Mediated Interaction of Tubulin with VDAC Revealed by Neutron Reflectometry

A preponderance of evidence suggests that the mitochondrial outer membrane (MOM) binds dimeric tubulin, which blocks the flux of metabolites through VDAC, the major MOM channel, by inserting its negatively charged, disordered C-terminal tail into the VDAC pore. The discovery of this novel regulatory mechanism of mitochondrial respiration has raised several fundamental questions, including how tubulin binds to the MOM surface, whether its membrane-bound conformation is consistent with the accessibility of its C-termini to the membrane surface, and what role mitochondrial lipids assume in mediating the tubulin-VDAC interaction. Here we report neutron reflectivity (NR) studies on tubulin-coated and VDAC-containing biomimetic membranes. In combination with molecular dynamics simulations, these reveal, first, that dimeric tubulin is anchored to the membrane surface by an amphipathic a-helix of the a-tubulin subunit in an orientation that presents the C-termini of both tubulin subunits to the membrane surface. The structural model is supported by electrophysiological evidence using recombinant tubulin constructs in which C-terminal tails bound to either subunit are individually observed in a VDAC nanopore. Second, we demonstrate using NR that surface-immobilized VDAC can be reconstituted into a complete lipid membrane, thus creating a “protein-tethered bilayer lipid membrane” (ptBLM) over a large sample area. The ptBLM architecture allows dynamic changes of the solution environment, while NR reveals the corresponding structural response of the protein and its lipid environment. This allows us to experimentally observe the effects of pH-induced changes in VDAC's structure on the local lipid environment. Molecular dynamics simulations are in good agreement with NR experiments. We speculate that local curvature or lipid packing stress induced by membrane-embedded VDAC and its structural dynamics serves to localize cytosolic regulators such as tubulin to the vicinity of the channel.