Differences In pH Dependence Research Articles

Unraveling the Intertwined Effect of pH on Helicobacter pylori Motility and the Microrheology of the Mucin-Based Medium It Swims in.

The gastric pathogen, Helicobacter pylori bacteria have to swim across a pH gradient from 2 to 7 in the mucus layer to colonize the gastric epithelium. Previous studies from our group have shown that porcine gastric mucin (PGM) gels at an acidic pH < 4, and H. pylori bacteria are unable to swim in the gel, although their flagella rotate. Changing pH impacts both the rheological properties of gastric mucin and also influences the proton (H+)-pumped flagellar motors of H. pylori as well as their anti-pH sensing receptors. To unravel these intertwined effects of acidic pH on both the viscoelastic properties of the mucin-based mucus as well as the flagellar motors and chemo-receptors of the bacterium, we compared the motility of H. pylori in PGM with that in Brucella broth (BB10) at different pH values using phase contrast microscopy to track the motion of the bacteria. The results show that the distribution of swimming speeds and other characteristics of the bacteria trajectories exhibit pH-dependent differences in both media. The swimming speed exhibits a peak at pH 4 in BB10, and a less pronounced peak at a higher pH of 5 in PGM. At all pH values, the bacteria swam faster and had a longer net displacement in BB10 compared to PGM. While the bacteria were stuck in PGM gels at pH < 4, they swam at these acidic pH values in BB10, although with reduced speed. Decreasing pH leads to a decreased fraction of motile bacteria, with a decreased contribution of the faster swimmers to the distributions of speeds and net displacement of trajectories. The body rotation rate is weakly dependent on pH in BB10, whereas in PGM bacteria that are immobilized in the low pH gel are capable of mechano-sensing and rotate faster. Bacteria can be stuck in the gel in various ways, including the flagella getting entangled in the fibers of the gel or the cell body being stuck to the gel. Our results show that in BB10, swimming is optimized at pH4, reflecting the combined effects of pH sensing by anti-pH tactic receptors and impact on H+ pumping of flagellar motors, while the increase in viscosity of PGM with decreasing pH and gelation below pH 4 lead to further reduction in swimming speed, with optimal swimming at pH 5 and immobilization of bacteria below pH 4.

A pH-sensitive switch activates virulence in Salmonella.

The transcriptional regulator SsrB acts as a switch between virulent and biofilm lifestyles of non-typhoidal Salmonella enterica serovar Typhimurium. During infection, phosphorylated SsrB activates genes on Salmonella Pathogenicity Island-2 (SPI-2) essential for survival and replication within the macrophage. Low pH inside the vacuole is a key inducer of expression and SsrB activation. Previous studies demonstrated an increase in SsrB protein levels and DNA-binding affinity at low pH; the molecular basis was unknown (Liew et al., 2019). This study elucidates its underlying mechanism and in vivo significance. Employing single-molecule and transcriptional assays, we report that the SsrB DNA-binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, His12, a conserved residue in the receiver domain confers pH sensitivity to SsrB allosterically. Acid-dependent DNA binding was highly cooperative, suggesting a new configuration of SsrB oligomers at SPI-2-dependent promoters. His12 also plays a role in SsrB phosphorylation; substituting His12 reduced phosphorylation at neutral pH and abolished pH-dependent differences. Failure to flip the switch in SsrB renders Salmonella avirulent and represents a potential means of controlling virulence.

The Implications of Coupling an Electron Transfer Mediated Oxidation with a Proton Coupled Electron Transfer Reduction in Hybrid Water Electrolysis.

Invited for this month's cover are the groups of Menny Shalom at the Ben-Gurion University of the Negev, Israel and Dr. Biswajit Mondal at Indian Institute of Technology Gandhinagar, India. The image shows the connection between two half-cells: an electron transfer-mediated [(2,2,6,6-tetramethyl-1-piperidin-1-yl)oxyl] (TEMPO)-catalyzed benzylamine oxidation at the anode and a proton-coupled electron transfer at the cathode for hydrogen generation. The difference in pH dependence of the anodic and cathodic processes enables hybrid water electrolysis at low cell potential (∼1.0 V) by adjusting only the pH value of the electrolytic medium. The Research Article itself is available at 10.1002/cssc.202202271.

Front Cover: The Implications of Coupling an Electron Transfer Mediated Oxidation with a Proton Coupled Electron Transfer Reduction in Hybrid Water Electrolysis (ChemSusChem 10/2023)

The Front Cover shows the combination of an electron transfer mediated anode for [(2,2,6,6-tetramethyl-1-piperidin-1-yl)oxyl] (TEMPO)-catalysed benzylamine oxidation to valuable chemicals and proton-coupled electron transfer process at the cathode for hydrogen generation from water. The difference in pH dependence of the anodic and cathodic processes enables hybrid water electrolysis at low cell potential (∼1.0 V) by adjusting only the pH value of the electrolytic medium. Product selectivity and rate of the anodic reaction can also be controlled by adjusting the pH, keeping the amount of hydrogen production unaffected. More information can be found in the Research Article by B. Mondal et al.

Physico-chemical characterization of aspirated and simulated human gastric fluids to study their influence on the intrinsic dissolution rate of cinnarizine

To elucidate the critical parameters affecting drug dissolution in the human stomach, the intrinsic dissolution rate (IDR) of cinnarizine was determined in aspirated and simulated human gastric fluids (HGF). Fasted aspirated HGF (aspHGF) was collected from 23 healthy volunteers during a gastroscopic examination. Hydrochloric acid (HCl) pH 1.2, fasted state simulated gastric fluid (FaSSGF), and simulated human gastric fluid (simHGF) developed to have rheological, and physico-chemical properties similar to aspHGF, were used as simulated HGFs. The IDR of cinnarizine was significantly higher in HCl pH 1.2 (952 ± 27 µg/(cm2·min)) than in FaSSGF pH 1.6 (444 ± 7 µg/(cm2·min)), and simHGF pH 2.5 (49 ± 5 µg/(cm2·min)) due to the pH dependent drug solubility and viscosity differences of the three simulated HGFs. The shear thinning behavior of aspHGF had a significant impact on the IDR of cinnarizine, indicating that the use of FaSSGF, with viscosity similar to water, to evaluate gastric drug dissolution, might overestimate the IDR by a factor of 100–10.000, compared to the non-Newtonian, more viscous, fluids in the human stomach. The developed simHGF simulated the viscosity of the gastric fluids, as well as the IDR of the model drug, making it a very promising medium to study gastric drug dissolution in vitro.

Self-assembling, pH-responsive nanoflowers for inhibiting PAD4 and neutrophil extracellular trap formation and improving the tumor immune microenvironment

Self-assembling carrier-free nanodrugs are attractive agents because they accumulate at tumor by an enhanced permeability and retention (EPR) effect without introduction of inactive substances, and some nanodrugs can alter the immune environment. We synthesized a peptidyl arginine deiminase 4 (PAD4) molecular inhibitor, ZD-E-1M. It could self-assembled into nanodrug ZD-E-1. Using confocal laser scanning microscopy, we observed its cellular colocalization, PAD4 activity and neutrophil extracellular traps (NETs) formation. The populations of immune cells and expression of immune-related proteins were determined by single-cell mass cytometry. ZD-E-1 formed nanoflowers in an acidic environment, whereas it formed nanospheres at pH 7.4. Accumulation of ZD-E-1 at tumor was pH-responsive because of its pH-dependent differences in the size and shape. It could enter the nucleus and bind to PAD4 to prolong the intracellular retention time. In mice, ZD-E-1 inhibited tumor growth and metastasis by inhibiting PAD4 activity and NETs formation. Besides, ZD-E-1 could regulate the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins like LAG3 were suppressed, while IFN-γ and TNF-α as stimulators of tumor immune response were upregulated. Overall, ZD-E-1 is a self-assembling carrier-free nanodrug that responds to pH, inhibits PAD4 activity, blocks neutrophil extracellular traps formation, and improves the tumor immune microenvironment.

Adsorption of Phosphorus Oxyanions at the FeOOH(goethite)/Water Interface: The Importance of Basicity.

PIII-containing H-phosphonate (HPO32-) and its monoethyl ester (fosetyl), essential pesticides for control of oomycete and fungal pathogens, are among the few pesticides transported by both xylem and phloem, making application as folia spray, soil spray, and trunk injection equally effective. To understand bioavailability and efficacy within soils, knowledge of adsorption to soil minerals is important. FeOOH(goethite) is often selected as an archetypal mineral surface. In the present work, H-phosphonate (with pKa values of 1.5 and 6.78) adsorption onto FeOOH is nearly complete below pH 6 and decreases to negligible amounts by pH 11, following an S-shaped curve. Fosetyl (pKa: 0.9), in contrast, does not adsorb to any significant extent, regardless of pH. To place these observations in context, adsorption of six other phosphorus oxyanions was investigated, and fitted using a CD-MUSIC model. Phosphate defines a similar S-shaped curve but adsorbs more strongly than H-phosphonate. Despite moderate differences in basicity, pH dependence and extents of adsorption for the four additional diprotic oxyanions methylphosphonate (pKas: 2.40, 8.00), benzylphosphonate (2.24, 7.93), phenylphosphonate (1.9, 7.47), and phenyl phosphate (1.1, 6.28) are quite similar to those of H-phosphonate. As with fosetyl, the other low pKa monoprotic oxyanion in our study, phenylphosphinate (pKa: 1.75), does not adsorb. Basicity, that is, pKa, is revealed to be the principal determinant of extents of adsorption.

PH effects on the electrochemical reduction of CO(2) towards C2 products on stepped copper

We present a microkinetic model for CO(2) reduction (CO(2)R) on Cu(211) towards C2 products, based on energetics estimated from an explicit solvent model. We show that the differences in both Tafel slopes and pH dependence for C1 vs C2 activity arise from differences in their multi-step mechanisms. We find the depletion in C2 products observed at high overpotential and high pH to arise from the 2nd order dependence of C-C coupling on CO coverage, which decreases due to competition from the C1 pathway. We further demonstrate that CO(2) reduction at a fixed pH yield similar activities, due to the facile kinetics for CO2 reduction to CO on Cu, which suggests C2 products to be favored for CO2R under alkaline conditions. The mechanistic insights of this work elucidate how reaction conditions can lead to significant enhancements in selectivity and activity towards higher value C2 products.

Role of cationic carbosilane dendrons and metallic core of functionalized gold nanoparticles in their interaction with human serum albumin

Functionalization of gold nanoparticles by different chemical groups is an important issue regarding the biomedical applications of such particles. Therefore we have analyzed the interaction between gold nanoparticles functionalized by carbosilane dendrons with human serum albumin at different pHs, and in the presence of the protein unfolding agent, guanidine hydrochloride, using circular dichroism, zeta-potential and fluorescence quenching. The effect of a nanoparticle dendronization and pure dendrons on the immunoreactivity of albumin was estimated using ELISA. In addition, the tool to estimate the binding capacity of dendronized gold nanoparticles using a hydrophobic fluorescent probe 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid) was chosen. We concluded that the effect of a nanoparticle on the structure, immunochemical properties and unfolding of albumin significantly decreased with second and third generations dendrons attached. Differences in pH dependence of the interaction between nanoparticles, their dendrons and albumin showed several effects of the “dendritic corona” and the metallic part of nanoparticle on the protein. These interactions indicate changes in the immunoreactivity of the protein, whereas dendron coating per se had no effect. Thus, dendronization of gold nanoparticles helps to shield them from interactions with plasma proteins.

A computational assessment of pH-dependent differential interaction of T7 lysozyme with T7 RNA polymerase

BackgroundT7 lysozyme (T7L), also known as N-acetylmuramoyl-L-alanine amidase, is a T7 bacteriophage gene product. It involves two functions: It can cut amide bonds in the bacterial cell wall and interacts with T7 RNA polymerase (T7RNAP) as a part of transcription inhibition. In this study, with the help of molecular dynamics (MD) calculations and computational interaction studies, we investigated the effect of varying pH conditions on conformational flexibilities of T7L and their influence on T7RNAP -T7L interactions.ResultsFrom the MD studies of the T7L at three different pH strengths viz. 5, neutral and 7.9 it was observed that T7L structure at pH 5 exhibited less stable nature with more residue level fluctuations, decrease of secondary structural elements and less compactness as compared to its counterparts: neutral pH and pH 7.9. The T-pad analysis of the MD trajectories identified local fluctuations in few residues that influenced the conformational differences in three pH strengths. From the docking of the minimum energy representative structures of T7L at different pH strengths (obtained from the free energy landscape analysis) with T7RNAP structures at same pH strengths, we saw strong interaction patterns at pH 7.9 and pH 5. The MD analysis of these complexes also confirmed the observations of docking study. From the combined in silico studies, it was observed that there are conformational changes in N-terminal and near helix 1 of T7L at different pH strengths, which are involved in the T7RNAP interaction, thereby varying the interaction pattern.ConclusionSince T7L has been used for developing novel therapeutics and T7RNAP one of the most biologically useful protein in both in-vitro and in vivo experiments, this in silico study of pH dependent conformational differences in T7L and the differential interaction with T7RNAP at different pH can provide a significant insight into the structural investigations on T7L and T7RNAP in varying pH environments.

Resonance Raman spectroscopy reveals pH-dependent active site structural changes of lactoperoxidase compound 0 and its ferryl heme O-O bond cleavage products.

The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this "stored" proton to the hydroperoxo fragment, it rapidly undergoes O-O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe-O-O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O-O bond-cleavage product, whose ν(Fe═O) frequencies reflect substantially different Fe═O bond strengths. In the process of conducting these studies, rR structural characterization of the dioxygen adduct of LPO, commonly called Compound III, has also been completed, demonstrating a substantial difference in the strengths of the Fe-O linkage of the Fe-O-O fragment under acidic and alkaline conditions, an effect most reasonably attributed to a corresponding weakening of the trans-axial histidyl imidazole linkage at lower pH. Collectively, these new results provide important insight into the impact of pH on the disposition of the key Fe-O-O and Fe═O fragments of intermediates that arise in the enzymatic cycles of LPO, other mammalian peroxidases, and related proteins.

Effect of pH on the activity of proteinases in intestinal mucosa, chyme, and microbiota of fish from the Cuciurgan reservoir

Proteolytic activities of the intestinal mucosa, chyme, and enteral microbiota have been studied in a wide range of pH values in five fish species from the Cuciurgan reservoir (Moldova). Differences in pH dependence of the intestinal proteinase activity of fish are determined by their feeding type. The maximum activity of proteinases is found in the pumpkinseed Lepomis gibbosus. The minimum activity of proteinases has been demonstrated by the zander Zander lucioperca. The pH optimum of the mucosa and chyme in all fish species (except for the European perch Perca fluviatilis) is 10. The pH optimum of the intestinal microflora varies from 6.0 (in the common carp Cyprinus carpio) to 10 (in the crucian carp Carassius carassius), whereas that in the perch from the Cuciurgan and Rybinsk reservoirs is 7. The majority of fish species, mostly Zander lucioperca and Lepomis gibbosus, are characterized by high proteniase activity of the microbiota, in the pH ranging from 6 to 9. It is assumed that proteinases in the enteral microbiota of fish are able to make up for the relatively low activity of those synthesized by their digestive system in the range of low pH values.

Effect of habitat conditions on proteinase and glycosidase activities in the gut microbiota of crucian carp in a wide range of pH

Significant differences in pH dependence of proteolytic and amylolytic enzyme activities in the gut microbiota between crucian carps from different habitats (the Dniester River and Kuchurgan Reservoir) have been revealed for the first time. The proteolytic activity in fish from these water bodies reaches a peak at pH values varying from 7 to 10, while the peak of amylolytic activity is consistently observed at pH 7.0. Both proteolytic and amylolytic activities are higher in fish from the Kuchurgan Reservoir (characterized by increased trophicity and high abundance of bacterioplankton) than in fish from the Dniester. For comparison, the effect of pH on proteinase and glycosidase activities in the gut mucosa and chyme of the same fish has been studied.

Q-Site Occupancy Defines Heme Heterogeneity in Escherichia coli Nitrate Reductase A (NarGHI)

The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme bD EPR signal arises from the differential occupancy of the Q-site. In agreement with this, the heterogeneity is dependent upon growth conditions but is still apparent when NarGHI is expressed in a strain lacking cardiolipin. Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A and NarI-K86A, and is collapsible by the binding of inhibitors. We found that the two main gz components of heme bD exhibit differences in reduction potential and pH dependence, which we posit is due to differential Q-site occupancy. Specifically, in a quinone-bound state, heme bD exhibits an Em,8 of -35 mV and a pH dependence of -40 mV pH(-1). In the quinone-free state, however, heme bD titrates with an Em,8 of +25 mV and a pH dependence of -59 mV pH(-1). We hypothesize that quinone binding modulates the electrochemical properties of heme bD as well as its EPR properties.

Structural changes in the C-terminal regulatory region of the Na+/H+ exchanger mediate phosphorylation induced regulation

The Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane pH regulatory protein that removes one intracellular H+ in exchange for an extracellular Na+. NHE1 is regulated by phosphorylation of the cytoplasmic regulatory region and amino acids Ser770 and Ser771 of the regulatory domain are necessary for activation by sustained intracellular acidosis. The phosphomimetic mutations (S770D/S771D) resulted in an activated form of the protein. Immunoprecipitation of full length NHE1 protein showed that the phosphomimetic mutant had increased sensitivity to digestion with trypsin indicating a conformational change. Tryptic digestion of purified C-terminal regulatory region showed that the S770D/S771D mutation altered sensitivity to trypsin digestion. Wild type and phosphomimetic purified C-terminal region (577–815) of human NHE1 were compared and tryptophan fluorescence indicated that there were pH-dependent differences in the conformation of the proteins. Native polyacrylamide gel electrophoresis demonstrated that the phosphomimetic mutant had a more compact structure. Bottom-up hydrogen/deuterium exchange mass spectrometry demonstrated that a peptide fragment containing the phosphomimetic mutations became strongly stabilized relative to the wild type protein. Overall, the results suggested that phosphorylation of S770/S771 changes the conformation of the C-terminal regulatory region in a pH-dependent manner, resulting in a more compact region that affects NHE1 activity. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

Sensory characteristics and functionality of sonicated whey

This research employed power ultrasound (US) on a whey suspension containing 28.2% solids (10% total protein) and characterized the US treated whey with respect to descriptive sensory evaluation, thermal stability, particle size, emulsification activity, and viscosity. The sensory attributes of sonicated and control whey were determined at three pH levels (3.5, 4.5 and 7.5) with and without nose clips using a trained panel. Nose clips were used to avoid the detection of aromatics and to evaluate whey quality in terms of taste only. There were pH dependent differences in 7 of 21 attributes tested with nose clips and there were no significant differences between the control and US samples when comparing the same pH levels, with the exception of cardboard which was found at a higher level in the pH 7.5 control sample than in the US pH 7.5 sample. There were 9 pH dependent differences in attributes when tested without nose clips and there were no significant differences between the control and US samples of the same pH, with the exception of cardboard being higher in the control pH 4.5 sample, and malty being higher in the control pH 7.5 sample compared to their respective US samples. Thermal stability was monitored via transmission of samples at 600nm and transmissions of US and control whey were significantly different based on temperature (72 or 85°C), concentration (5 or 10% solids) and pH (3.5, 4.5 and 7.5). Transmissions were higher in all US samples compared to the controls indicating less turbidity in US samples. Transmissions were higher in samples with the lower heating treatment of 72°C, at pH 7.5, and at 5% solids. US whey resulted in a more stable emulsion, lower particle size, and a higher viscosity compared to control whey.

Fluorescence Studies of gold(III)-Norfloxacin Complexes in Aqueous Solutions

Formation of gold(III) complexes with the synthetic antibiotic norfloxacin (NF) was investigated in aqueous solution at pH 4.0, 7.5 and 10.6, with the ligand in cationic, zwitterionic and anionic forms, respectively. UV-Visible spectroscopy, steady state and time-resolved fluorometry were used to characterize the complexes. Binding sites, association constants and fluorescence lifetimes of the complexes were obtained. Au(3+) binding to zwitterionic NF produced a fluorescence decrease and a small red shift. Fluorescence changes as a function of Au(3+) concentration were fitted using a one-site binding model and the association constant was obtained, K(bzw) = 1.7 X 10⁵ M⁻¹. The association of Au(3+) with cationic NF was much weaker, the obtained binding constant being K(bcat) = 2.4 X 10³ M⁻¹. The Au(3+) binding site for these species involves the carboxyl group, in agreement with a much stronger association of the cation with the carboxylate anion than with the neutral acid. Association of Au(3+) with nonfluorescent anionic NF presented a clear evidence of two binding sites. The highest affinity site is the unprotonated piperazinyl group with K(bpip) ≥ 5 X 10⁷ M⁻¹ , and the low affinity site includes the carboxylate anion. The results point out to important pH dependent differences in complex formation between transition metal ions and fluoroquinolones, leading to different binding sites and association constants that change by several orders of magnitude.

A Comparison of the Higher Order Harmonic Components Derived from Large-Amplitude Fourier Transformed ac Voltammetry of Myoglobin and Heme in DDAB Films at a Pyrolytic Graphite Electrode

A debate as to whether heme remains bound or is released in myoglobin molecules incorporated into a didodecyldimethylammonium bromide (DDAB) film adhered to a pyrolytic graphite electrode has prompted a comparison of their electrochemistry by the highly sensitive large-amplitude Fourier transformed ac voltammetric method. The accessibility of third, fourth, and higher harmonic components that are devoid of background current and the enhanced resolution relative to that available in dc voltammetry have allowed a detailed comparison of the Fe(III)/Fe(II) and Fe(II)/Fe(I) redox processes of myoglobin and heme molecules to be undertaken as a function of buffer composition and pH and in the presence and absence of NaBr in the buffer and/or film. Under most conditions examined, only very subtle differences, in the Fe(III)/Fe(II) process were found, implying this process cannot be used to indicate the intactness or otherwise of myoglobin in myoglobin-DDAB films. In contrast, higher order ac harmonics obtained from myoglobin-DDAB and heme-DDAB films reveal pH dependent differences with respect to the Fe(II)/Fe(I) couple. Analysis of the ac harmonics, and with the hypothesis that the Fe(II)/Fe(I) process reflects the myoglobin state, suggests that the majority of the iron heme is released from myoglobin-DDAB (pH 5.0, no NaBr) films in contact with pH 5.0 (0.1 M sodium acetate) buffer solution devoid of or containing NaBr. However, myoglobin films prepared with pH 5.0 buffer containing NaBr shows significant difference in the higher harmonic shapes and midpoint potentials in the Fe(II)/Fe(I) process relative to the case when heme molecules are used, although as noted in other studies, a significant fraction of the Mb is rendered electroinactive in the presence of NaBr. The voltammetric responses of myoglobin and heme-DDAB (pH 5.0) films in contact with pH 7.0 (0.1 M) phosphate buffer solution also exhibit significant differences in the Fe(II)/Fe(I) redox couple in the higher harmonics in contrast to a report [de Groot, M.T.; Merkx, M.; Koper, M. T. M. J. Am. Chem. Soc. 2005, 127, 16224] that claimed identical midpoint potentials apply to both films under conditions of dc cyclic voltammetry. The FT-ac voltammetric data therefore suggest that a substantial fraction of myoglobin in myoglobin-DDAB (pH 5.0) films in contact with pH 7.0 phosphate buffer solution remains intact. No evidence of a catalytic effect that enhanced the released of heme from myoglobin was found at the pyrolytic graphite electrode surface. In summary, higher harmonic ac voltammetric data indicate that the Fe(II)/Fe(I) process but not the Fe(III)/Fe(II) reflects the state of myoglobin in DDAB films. On this basis, films prepared at pH 5.0 should include NaBr, or else films should be prepared at neutral pH to achieve films with myoglobin remains in its intact near native state when a myoglobin-DDAB film is confined to a graphite electrode surface. Otherwise, the release of heme in myoglobin molecules incorporated into a DDAB film is likely to be a dominant reaction pathway.

Role of Ribosomal Protein L27 in Peptidyl Transfer

The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T. thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward p K a shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.

Solid-State 19F NMR Spectroscopy Reveals That Trp41 Participates in the Gating Mechanism of the M2 Proton Channel of Influenza A Virus

The integral membrane protein M2 of influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH. The side chain of His37 in the transmembrane alpha-helix is known to play an important role in the pH activation of the proton channel. It has also been suggested that Trp41, which is located in an adjacent turn of the helix, forms part of the gating mechanism. Here, a synthetic 25-residue peptide containing the M2 transmembrane domain was labeled with 6F-Trp41 and studied in lipid membranes by solid-state 19F NMR. We monitored the pH-dependent differences in the 19F dipolar couplings and motionally narrowed chemical shift anisotropies of this 6F-Trp41 residue, and we discuss the pH activation mechanism of the H+ channel. At pH 8.0, the structural parameters implicate an inactivated state, while at pH 5.3 the tryptophan conformation represents the activated state. With the aid of COSMOS force field simulations, we have obtained new side-chain torsion angles for Trp41 in the inactivated state (chi1 = -100 degrees +/- 10 degrees , chi2 = +110 degrees +/- 10 degrees ), and we predict a most probable activated state with chi1 = -50 degrees +/- 10 degrees and chi2 = +115 degrees +/- 10 degrees . We have also validated the torsion angles of His37 in the inactivated state as chi1 = -175 degrees +/- 10 degrees and chi2 = -170 degrees +/- 10 degrees .