Protons In Response Research Articles

Site-directed mutagenesis at the Glu78 in Ec-NhaA transporter impacting ion exchange: a biophysical study.

Na+/H+ antiporters facilitate the exchange of Na+ for H+ across the cytoplasmic membrane in prokaryotic and eukaryotic cells. These transporters are crucial to maintain the homeostasis of sodium ions, consequently pH, and volume of the cells. Therefore, sodium/proton antiporters are considered promising therapeutic targets in humans. The Na+/H+ antiporter in Escherichia coli (Ec-NhaA), a prototype of cation-proton antiporter (CPA) family, transports two protons and one sodium (or Li+) in opposite direction. Previous mutagenesis experiments on Ec-NhaA have proposed Asp164, Asp163, and Asp133 amino acids with the significant implication in functional and structural integrity and create site for ion-binding. However, the mechanism and the sites for the binding of the two protons remain unknown and controversial which could be critical for pH regulation. In this study, we have explored the role of Glu78 in the regulation of pH by Ec-NhaA. Although we have created various mutants, E78C has shown a considerable effect on the stoichiometry of NhaA and presented comparable phenotypes. The ITC experiment has shown the binding of ~ 5 protons in response to the transport of one lithium ion. The phenotype analysis on selective medium showed a significant expression compared to WT Ec-NhaA. This represents the importance of Glu78 in transporting the H+ across the membrane where a single mutation with Cys amino acid alters the number of H+ significantly maintaining the activity of the protein.

Preparation of colloidal nanoparticles PVA-PHEMA from hydrolysis of copolymers of PVAc-PHEMA as anticancer drug carriers

The novel pH-responsive polymer nanoparticles have been widely used for drug delivery and cancer therapy. The pH-sensitive nanoparticles include chemical structures that can accept or donate protons in response to an environmental pH change. Polybases which mostly contain alkaline groups such as amines and hydroxy, accept protons at low pH and are neutral at higher pH values. This study aimed to prepare pH-sensitive colloidal amphiphilic poly(vinyl alcohol-2-hydroxyethyl methacrylate) (PVA-PHEMA) copolymers in cancer therapy applications. For this purpose, poly(vinyl acetate-2-hydroxyethyl methacrylate) (PVAc-PHEMA) copolymer nanoparticles were synthesized in different polymerization medium fractions from water and methanol and different monomer feed concentration. Then acetate groups were hydrolyzed, and the PHEMA-PVA nanoparticles were synthesized. The nanoparticles were further characterized using dynamic light scattering, Fourier transform infrared spectroscopy, scanning electron microscopy, and thermogravimetric analysis to identify the structural and morphological changes. The Methotrexate (MTX) was loaded into the nanoparticles, and drug release kinetics were evaluated. The results confirmed that PHEMA-PVA copolymeric nanoparticles could be favorably used in cancer therapy.

Stimuli-Induced Reversible Proton Transfer for Stimuli-Responsive Materials and Devices.

ConspectusStimuli-responsive materials have a great potential in various novel photoelectric devices, such as self-adaptive adjustment devices, intelligent detection, molecular computers with information storage capability, camouflage and anticounterfeiting display, various energy-saving displays, and others. However, progress in related areas has been relatively slow because of the lack of high-performance smart materials and the limitations of available reaction mechanisms currently. To address these problems fundamentally, new mechanisms need to be designed and developed, and learning from nature is an effective and intelligent method to achieve this long-awaited target, such as mimicking of proton transfer processes in nature at the molecular/supramolecular level. The stimuli-induced reversible proton transfer system is composed of materials that release or capture protons in response to stimuli and switch molecules that control color and/or fluorescence modulation by protons, and it is applied in stimuli-responsive materials and devices, including bistable electronic/electrochromic devices, electrofluorochromic devices, water-jet rewritable paper, visible-light-responsive rewritable paper, and mechanochromic materials.To help researchers gain deep insight into stimuli-induced reversible proton transfer, we attempted to summarize its reaction mechanism and design principle, and discuss strategies to design and prepare various related stimuli-responsive materials and devices. This Account discusses the different systems in which a color/fluorescence change is induced by the proton transfer process under various stimuli, including electric field, water, light, heat, and stress. Relative very promising applications as well as their performance especially for energy-saving and environmentally friendly devices are then summarized, such as energy-saving bistable electrochromic devices, water-jet rewritable paper, and visible-light-responsive rewritable paper. Meanwhile, we focus on the key influence factors and useful additives for improving the device's performance. At last, challenges and bottlenecks faced by stimuli-responsive materials and devices based on the mechanism of reversible proton transfer are proposed. Moreover, we put forward some suggestions on solving these limitations.These exciting results reveal that smart materials based on the mechanism of proton transfer are extremely attractive and possess great potential in the next generation of energy and resource saving and environmental protection display. We hope that this Account further prospers the field of intelligent stimuli-responsive discoloration materials and next-generation green displays.

Two‐Dimensional Hybrid Particle‐in‐Cell Simulations of Magnetosonic Waves in the Dipole Magnetic Field: On a Constant L‐Shell

AbstractTwo‐dimensional hybrid particle‐in‐cell (PIC) simulations are carried out on a constant L‐shell (or drift shell) surface of the dipole magnetic field to investigate the generation process of near‐equatorial fast magnetosonic waves (a.k.a equatorial noise; MSWs hereafter) in the inner magnetosphere. The simulation domain on a constant L‐shell surface adopted here allows wave propagation and growth in the azimuthal direction (as well as along the field line) and is motivated by the observations that MSWs propagate preferentially in the azimuthal direction in the source region. Furthermore, the equatorial ring‐like proton distribution used to drive MSWs in the present study is (realistically) weakly anisotropic. Consequently, the ring‐like velocity distribution projected along the field line by Liouville's theorem extends to rather high latitude, and linear instability analysis using the local plasma conditions predicts substantial MSW growth up to ±27° latitude. In the simulations, however, the MSW intensity maximizes near the equator and decreases quasi‐exponentially with latitude. Further analysis reveals that the stronger equatorward refraction at higher latitude due to the larger gradient of the dipole magnetic field strength prevents off‐equatorial MSWs from growing continuously, whereas MSWs of equatorial origin experience little refraction and can fully grow. Furthermore, the simulated MSWs exhibit a rather complex wave field structure varying with latitude, and the scattering of energetic ring‐like protons in response to MSW excitation occurs faster than the bounce period of those protons so that they do not necessarily follow Liouville's theorem during MSW excitation.

Calcineurin homologous protein isoform 2 supports tumor survival via the sodium hydrogen exchanger isoform 1 in non-small cell lung cancer.

Maintaining intracellular pH is crucial for preserving healthy cellular behavior and, when dysregulated, results in increased proliferation, migration, and invasion. The Na+/H+ exchanger isoform 1 is a highly regulated transmembrane antiporter that maintains pH homeostasis by exporting protons in response to intra- and extracellular signals. Activation of Na+/H+ exchanger isoform 1 is exquisitely regulated by the extracellular environment and protein cofactors, including calcineurin B homologous proteins 1 and 2. While Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 1 are ubiquitously expressed, calcineurin B homologous protein 2 shows tissue-specific expression and upregulation in a variety of cancer cells. In addition, calcineurin B homologous protein 2 expression is modulated by tumorigenic extracellular conditions like low nutrients. To understand the role of calcineurin B homologous protein 2 in tumorigenesis and survival in lung cancer, we surveyed existing databases and formed a comprehensive report of Na+/H+ exchanger isoform 1, calcineurin B homologous protein 1, and calcineurin B homologous protein 2 expression in diseased and non-diseased tissues. We show that calcineurin B homologous protein 2 is upregulated during oncogenesis in many adeno and squamous carcinomas. To understand the functional role of calcineurin B homologous protein 2 upregulation, we evaluated the effect of Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 2 depletion on cellular function during cancer progression in situ. Here, we show that calcineurin B homologous protein 2 functions through Na+/H+ exchanger isoform 1 to effect cell proliferation, cell migration, steady-state pHi, and anchorage-independent tumor growth. Finally, we present evidence that calcineurin B homologous protein 2 depletion in vivo has potential to reduce tumor burden in a xenograft model. Together, these data support the tumor-promoting potential of aberrant calcineurin B homologous protein 2 expression and position calcineurin B homologous protein 2 as a potential therapeutic target for the treatment of non-small cell lung cancer.

Design and synthesis of fluorescent shell functionalized polymer micelles for biomedical application

pH‐sensitive polymers can be defined as polyelectrolytes that include in their structure weak acidic or basic groups that either accept or release protons in response to a change in the environmental pH. This work summarizes the design, synthesis, and potential applications of pH‐responsive fluorescent copolymers in the biomedical field. This was achieved using atom transfer radical polymerization (ATRP) of tert‐butyl acrylate using a CuBr/N,N,N′,N″N″‐pentamethyldiethylenetriamine catalyst system in conjunction with an alkyl bromide as the initiator. Well‐defined macroinitiators based on poly(tert‐butyl acrylate) with narrow molecular weight distributions were obtained by the addition of an appropriate solvent system in order to create a homogeneous catalytic system. The addition of n‐butyl acrylate as a second building block in order to create well‐defined poly(tert‐butyl acrylate)‐b‐poly(n‐butyl acrylate) block copolymers (PtBA‐b‐PnBA) followed by chemical modification of the block copolymers and functionalization with an appropriate fluorescent compound are the basis for the preparation of well‐defined fluorescent pH‐sensitive micelles. Thus, prepared water soluble nanosized pH‐sensitive micelles consisting of hydrophobic poly(n‐butyl acrylate) core and hydrophilic polyacrylic acid shell decorated with an appropriate fluorescent compound determined their potential applications of these systems in the field of biomedicine as biosensors, controlled drug delivery systems, and so on. In this respect, the cell viability and internalization of the polymer micelles were studied.

Interfacial and Nanoconfinement Effects Decrease the Excited-State Acidity of Polymer-Bound Photoacids

Summary Photo-initiated ion transport on the nanoscale is relevant to various fields including energy conversion, neuron triggering, and biomimetic processes. To study this phenomenon, we synthesized and evaluated photoacid-modified polymers, which generate hydrated protons in response to visible-light excitation. Pyrenol photoacid dye molecules were covalently bonded within conical nanopores that had been track etched in poly(ethylene terephthalate). The data suggest that ∼90% of the nanopore surfaces were modified with photoacids and that photoacids were, on average, bound through three sulfonamide groups. In comparison to photoacids dissolved in an aqueous solution, photoacids bound specifically to the nanoporous tip region were, on average, weaker acids in their ground and excited states, which we presume is due to differences in surface potential or solvation environment in the confined nanopores. These results suggest that ion transport initiated by nanoconfined photoacids will require careful molecular engineering to enable efficient light-to-ionic energy conversion.

A pH‐responsive self‐fluorescent polymeric micelle as a potential optical imaging probe

A series of methoxypolyethylene glycol‐terminated self‐fluorescent polyurethane multi‐block copolymers with excellent pH‐responsivity, self‐fluorescence, and biocompatibility are designed and synthesized. In our design, 1, 4‐bis (hydroxyethyl) piperazine is chosen as a pH‐responsive segment which can donate or accept protons in response to the change of environmental pH, and fluorescein isothiocyanate is used as a fluorescent dye conjugated into the micelles to offer self‐fluorescence. The chemical structure of the polyurethane multi‐block copolymers is characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The results of the acid‐based titration, the fluorescence spectrometry, and the ultraviolet visible spectroscopy indicate that the polyurethane multi‐block copolymers own an excellent pH‐buffering capacity responded to the change of pH values and the favorable self‐fluorescence property in an aqueous solution. And the ultraviolet absorption peaks of samples are strengthened with increasing of pH values, indicating that methoxypolyethylene glycol‐terminated self‐fluorescent polyurethane multi‐block copolymer can be a pH‐dependent fluorescent probe in a broad pH range. In addition, the in vitro cytotoxicity test showed that the polyurethane multi‐block copolymer has low cytotoxicity and good biocompatibility, which make it a promising nanoplatform for molecular imaging, diagnosis, and treatment of disease.

Insight into the Role of the Hv1 C-Terminal Domain in Dimer Stabilization.

The voltage-gated proton-selective channel (Hv1) conducts protons in response to changes in membrane potential. The Hv1 protein forms dimers in the membrane. Crystal structures of Hv1 channels have revealed that the primary contacts between the two monomers are in the C-terminal domain (CTD), which forms a coiled-coil structure. The role of Hv1-CTD in channel assembly and activity is not fully understood. Here, molecular dynamics (MD) simulations of full-length and truncated CTD models of human and mouse Hv1 channels reveal a strong contribution of the CTD to the packing of the transmembrane domains. Simulations of the CTD models highlight four fundamental interactions of the key residues contributing to dimer stability. These include salt bridges, hydrophobic interactions, hydrogen bonds, and a disulfide bond across the dimer interface. At neutral pH, salt-bridge interactions increase dimer stability and the dimer becomes less stable at acidic pH. Hydrophobic core packing of the heptad pattern is important for stability, as shown by favorable nonpolar binding free energies rather than by electrostatic components. Moreover, free-energy calculations indicate that a more uniform hydrophobic core in the coiled-coil structure of the Hv1-NIN, a channel carrying the triple mutation M234N-N235I-V236N, leads to an increase in dimer stability with respect to the wild-type. A Cys disulfide bond has a strong impact on dimer stability by holding the dimer together and facilitating the interactions described above. These results are consistent with dissociative temperatures and energy barriers of dimer dissociation obtained from the temperature-accelerated MD.

Commentary: The Spinal Cord Has an Intrinsic System for the Control of pH.

Most physiological processes are sensitive to pH (i.e., hydrogen ion activity) and regulation of pH within tissues and body fluids represents a fundamental homeostatic process in living organisms. At the organismal level, acid-base balance is achieved by fast respiratory modulations of the partial pressure of CO2 in combination with slower transepithelial ion movements to accommodate excretion and retention of bicarbonate and protons in response to acid-base disturbances. At the cellular and subcellular levels, acid-base balance is achieved by ion exchange.

Characterization of a Novel Voltage-Sensing Protein

The discovery of Ci-VSP, a voltage-sensing phosphatase, revealed that S1-S4 domains can exist in proteins independent of an ion-conducting pore (Nature, 2005). The voltage-activated proton channel, Hv1, was subsequently discovered and shown to consist of a voltage-sensing domain that conducts protons in response to membrane depolarization (Nature, 2006). Through bioinformatic searches, we identified a protein that we named NVS (Novel Voltage Sensor). NVS contains 531 residues and consists of 3 parts: an S1-S4 domain, a 90 residue N-terminus and a 307 residue C-terminus, both of which are predicted to be intracellular. The most critical residues found in other S1-S4 domains are conserved in NVS, including 3 Arg and a Lys in the S4 helix, and 4 conserved acidic residues in S1-S3. Other than an S1-S4 domain, NVS contains no conserved domains that offer clues about its function. However, the C-terminus does contain a coiled-coil domain, several SH3 binding motifs and a region that has 30% identity with a Ras-GAP binding protein thought to regulate Ras signaling pathways. Here, we show that NVS traffics to the membrane as determined by surface biotinylation. Additionally, tissue distribution studies show expression of NVS in brain, heart, kidney, liver and testes. Within the brain, NVS is enriched in cerebellum and immunofluorescence studies on frozen tissue sections indicate that NVS localizes to pre-synaptic terminals of granule cells. Furthermore, the S4 helix of NVS is capable of sensing changes in membrane potential as revealed by transferring this region into Hv1. Our guiding hypothesis is that NVS functions as a voltage sensor that interacts with signaling proteins to provide intracellular pathways with information about voltage changes across the membrane. To this end, experiments are underway to identify interacting proteins for clues about the function of NVS.

Rhizosphere interactions between microorganisms and plants govern iron and phosphorus acquisition along the root axis – model and research methods

Iron and phosphorus availability is low in many soils; hence, microorganisms and plants have evolved mechanisms to acquire these nutrients by altering the chemical conditions that affect their solubility. In plants, this includes exudation of organic acid anions and acidification of the rhizosphere by release of protons in response to iron and phosphorus deficiency. Grasses (family Poaceae) and microorganisms further respond to Fe deficiency by production and release of specific chelators (phytosiderophores and siderophores, respectively) that complex Fe to enhance its diffusion to the cell surface. In the rhizosphere, the mutual demand for Fe and P results in competition between plants and microorganisms with the latter being more competitive due to their ability to decompose plant-derived chelators and their proximity to the root surface; however microbial competitiveness is strongly affected by carbon availability. On the other hand, plants are able to avoid direct competition with microorganisms due to the spatial and temporal variability in the amount and composition of exudates they release into the rhizosphere. In this review, we present a model of the interactions that occur between microorganisms and roots along the root axis, and discuss advantages and limitations of methods that can be used to study these interactions at nanometre to centimetre scales. Our analysis suggests mechanisms such as increasing turnover of microbial biomass or enhanced nutrient uptake capacity of mature root zones that may enhance plant competitiveness could be used to develop plant genotypes with enhanced efficiency in nutrient acquisition. Our model of interactions between plants and microorganisms in the rhizosphere will be useful for understanding the biogeochemistry of P and Fe and for enhancing the effectiveness of fertilization.

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Members of the CLC gene family either function as chloride channels or as anion/proton exchangers. The plant AtClC-a uses the pH gradient across the vacuolar membrane to accumulate the nutrient NO(3)(-) in this organelle. When AtClC-a was expressed in Xenopus oocytes, it mediated NO(3)(-)/H(+) exchange and less efficiently mediated Cl(-)/H(+) exchange. Mutating the "gating glutamate" Glu-203 to alanine resulted in an uncoupled anion conductance that was larger for Cl(-) than NO(3)(-). Replacing the "proton glutamate" Glu-270 by alanine abolished currents. These could be restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4 and ClC-5 mediate stoichiometrically coupled 2Cl(-)/H(+) exchange, their NO(3)(-) transport is largely uncoupled from protons. By contrast, the AtClC-a-mediated NO(3)(-) accumulation in plant vacuoles requires tight NO(3)(-)/H(+) coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this proline was mutated to serine (P160S), Cl(-)/H(+) exchange of AtClC-a proceeded as efficiently as NO(3)(-)/H(+) exchange, suggesting a role of this residue in NO(3)(-)/H(+) exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl(-)/H(+) exchanger gained efficient NO(3)(-)/H(+) coupling. When inserted into the model Torpedo chloride channel ClC-0, the equivalent mutation increased nitrate relative to chloride conductance. Hence, proline in the CLC pore signature sequence is important for NO(3)(-)/H(+) exchange and NO(3)(-) conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.

Cytosolic Delivery of Membrane-Impermeable Molecules in Dendritic Cells Using pH-Responsive Core−Shell Nanoparticles

Polycations that absorb protons in response to the acidification of endosomes can theoretically disrupt these vesicles via the "proton sponge" effect. To exploit this mechanism, we created nanoparticles with a segregated core-shell structure for efficient, noncytotoxic intracellular drug delivery. Cross-linked polymer nanoparticles were synthesized with a pH-responsive core and hydrophilic charged shell designed to disrupt endosomes and mediate drug/cell binding, respectively. By sequestering the relatively hydrophobic pH-responsive core component within a more hydrophilic pH-insensitive shell, nontoxic delivery of small molecules and proteins to the cytosol was achieved in dendritic cells, a key cell type of interest in the context of vaccines and immunotherapy.

The Roles of Acids and Bases in Enzyme Catalysis

Many organic reactions are catalyzed by strong acids or bases that protonate or deprotonate neutral reactants leading to reactive cations or anions that proceed to products. In enzyme reactions, only weak acids and bases are available to hydrogen bond to reactants and to transfer protons in response to developing charges. Understanding this difference goes a long way toward applying organic concepts to the mechanisms of bioorganic chemistry.

Fluorosensors for ammonia using rhodamines immobilized in plasticized poly(vinyl chloride) and in sol-gel; a comparative study

We report on fluorosensors for ammonia using rhodamines incorporated in plasticized poly(vinyl chloride) (PVC) and in sol-gel matrix. The response of rhodamine B is due to a change in its molecular structure. When exposed to ammonia, the rhodamine is converted into a colorless, non-fluorescent lactone. As a result, the decrease in fluorescence intensity is proportional to the concentration of ammonia. To improve sensitivity we make use of fluorescence energy transfer using donor-acceptor complexes. Ion pairs consisting of a fluorophore (rhodamine B or tetramethylrhodamine ethyl ester) and an absorber (bromophenol blue) were immobilized in PVC and in sol-gel. Response times of the optode layers are in the order of 2 to 8 min depending on the ammonia concentration and the matrix used. Limits of detection are as low as 0.1 μg ml −1 ammonia. The sol-gel is appropriate for use in sensor technology and comparable to PVC layers with respect to response mechanism and response time. Sol-gel based ammonia sensors are advantageous over PVC layers in their storage stability (≥6 months) and the relative insensitivity to protons in response to ammonia.

The Essential Arginine Residue at Position 210 in the a Subunit of the Escherichia coli ATP Synthase Can Be Transferred to Position 252 with Partial Retention of Activity

The substitution of arginine at position 210 in the alpha subunit of Escherichia coli F0F1-ATPase by either lysine or alanine causes dominance in complementation tests with a chromosomal c subunit mutation. Reversal of dominance was achieved for the alpha R210K mutation but not for the alpha R210A mutation by the presence of an aspartic acid residue at position 50 or at position 252 in the alpha subunit. It was concluded that position 210 in putative helix 4 of a previously proposed model of the alpha subunit is close to position 252 in putative helix 5 and to position 50 in putative helix 1. The juxtaposition of residues 252 and 210 was also indicated by the observation that the double mutant alpha R210Q/Q252R was partially functional. A revertant of the partially functional double mutant, isolated on succinate medium, was found to contain a third mutation resulting in Pro-204 in the alpha subunit being replaced by threonine. That the revertant phenotype was due to the alpha P204T change was confirmed by site-directed mutagenesis. ATP synthesis in the revertant strain was at near normal levels as judged by growth yield experiments, but the revertant strain was unable to pump protons in response to ATP hydrolysis.

Characterization of energetically functional inverted membrane vesicles from Corynebacterium glutamicum

We show that inverted membrane vesicles from Corynebacteriwn glutamicum, a Gram-positive bacterium, are able to generate and maintain an electrochemical gradient of protons in response to the addition of NADH. This result indicates that the respiratory chain is intact and that the vesicles are reasonably impermeable to protons. These membrane vesicles may be the starting point for in vitro translocation studies of proteins in Gram-positive bacteria.

Respiration-linked proton translocation coupled to anaerobic reduction of manganese(IV) and iron(III) in Shewanella putrefaciens MR-1

An oxidant pulse technique, with lactate as the electron donor, was used to study respiration-linked proton translocation in the manganese- and iron-reducing bacterium Shewanella putrefaciens MR-1. Cells grown anaerobically with fumarate or nitrate as the electron acceptor translocated protons in response to manganese (IV), fumarate, or oxygen. Cells grown anaerobically with fumarate also translocated protons in response to iron(III) and thiosulfate, whereas those grown with nitrate did not. Aerobically grown cells translocated protons only in response to oxygen. Proton translocation with all electron acceptors was abolished in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (20 microM) and was partially to completely inhibited by the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (50 microM).

Development of membrane-based biosensors: measurement of current from photocycling bacteriorhodopsin on patch clamp electrodes.

Our initial work toward developing membrane protein-based biosensors has involved use of bacteriorhodopsin (BR) as a model membrane protein. BR was incorporated into liposomes of a polymerizable lecithin, and was shown to pump protons in response to illumination both before and after polymerization of the lipids. In the work described in this paper, BR was first reincorporated in liposomes of asolectin by consonication with purple membrane. The liposomes, which sustained the function of the protein, were used to form a monolayer at the air-water interface. This monolayer was transferred as a bilayer onto patch electrode. When illuminated with a pulse of 514.5-nm light the lipid/protein patch produced a current spike into the pipette corresponding to events no later than the generation of the 412-nm intermediate, probably caused by pumping of protons across the patch membrane. The experiment demonstrates not only the extreme sensitivity of amperometric detection, but also a small tendency for membrane proteins to preferentially orient in this configuration.