pH-dependent Activity Research Articles

Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH⩽2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter.Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known “classical” Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved.

A tricarboxylated PtCl(terpyridine) derivative exhibiting pH-dependent photocatalytic activity for H₂ evolution from water.

The first negatively charged PtCl(tpy) (tpy = 2,2':6',2''-terpyridine) derivative, formulated as Na2[PtCl(tctpy)]·5H2O (tctpy = 2,2':6',2''-terpyridine-4,4',4''-tricarboxylate), was prepared, characterized, and investigated in detail for its activity as a single-component photocatalyst that drives water reduction to H2 in the presence of a sacrificial electron donor (EDTA). This compound was confirmed to exist in its fully deprotonated form [PtCl(tctpy)](2-) in aqueous media at pH > 4.4. Despite its dianionic character, [PtCl(tctpy)](2-) was found to form a specific adduct with anionic EDTA (i.e., YH2(2-) and YH(3-), where YH4 is a fully protonated form of EDTA), enabling reductive quenching of the triplet metal-to-ligand charge transfer excited state within the adduct, leading to subsequent electron transfer steps correlated with Pt(II)-catalyzed H2 evolution from water. Electrochemical studies also reveal that the compound exhibits a unique pH-dependent first reduction (i.e., tctpy-centered reduction), leading to our realization of the first example of a Pt(II)-based molecular system that photocatalyzes the H2 evolution reaction accompanied by a ligand-based proton-coupled electron transfer (PCET) process.

Crystal Structures of Streptococcus Pyogenes Cas2 Protein at Various pH Conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (cas) proteins provide RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids in archaea and bacteria. Cas2 protein is one of the two core Cas proteins are present in all types of CRISPR-Cas systems and is required for new spacer integration into CRISPR loci. Cas2 homologues from several CRISPR-Cas subtypes were characterized as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the X-ray crystal structures of Streptococcus pyogenes Cas2 protein at three different pHs (5.6, 6.5, and 7.5), and the results of its nuclease activity assay at varying pHs (6.0-9.0). While S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues suggest structural variability and the flexible nature of its putative hinge regions, supporting the supposition that conformational change is important for catalysis.

Effect of Histidine Rich Antimicrobial Peptides, Gad-1 and Gad-2, On Model Membranes at Different pH Values

Antimicrobial polypeptides (AMPs) are an important component of the innate immune system of many different organisms and are generally amphipathic and cationic in nature. Electrostatic interactions between positively charged residues of the AMP and negatively charged lipids in pathogen membranes are an important component of AMP mechanism and specificity. AMPs such as Gad-1 and Gad-2 that are rich in histidine, an amino acid that is uncharged at neutral pH and positively charged at slightly acidic pH, can thus exhibit pH dependent activity. 2H solid state NMR spectroscopy was used to study the effect of Gad-1 and Gad-2 on model membranes at different pH values. The addition of peptides to the model membrane system POPE/POPG-d31 (3:1) results in a change in the splitting of spectrum both at pH 7 and pH 5. Further investigation of peptide effects on lipid acyl chain motion were determined through calculations of orientational order parameter values (SCD). The order parameter profile demonstrated that Gad-1 causes more lipid acyl chain disordering than Gad-2, at both pH 7 and pH 5. These results are consistent with Gad-1's greater activity in assays for bacterial growth inhibition. On the other hand, while Gad-2 is more active at inhibiting bacterial growth at low pH than at neutral pH, its effect on lipid acyl chain disorder is the same at pH 7 and pH 5.

LexA protein of cyanobacterium Anabaena sp. strain PCC7120 exhibits in vitro pH-dependent and RecA-independent autoproteolytic activity

The LexA protein of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 exhibits a RecA-independent and alkaline pH-dependent autoproteolytic cleavage. The autoproteolytic cleavage of Anabaena LexA occurs at pH 8.5 and above, stimulated by the addition of Ca2+ and in the temperature range of 30–57°C. Mutational analysis of Anabaena LexA protein indicated that the cleavage occurred at the peptide bond between Ala-84 and Gly-85, and optimal cleavage required the presence of Ser-118 and Lys-159, as also observed for LexA protein of Escherichia coli. Cleavage of Anabaena LexA was affected upon deletion of three amino acids, 86GLI. These three amino acids are unique to all cyanobacterial LexA proteins predicted to be cleavable. The absence of RecA-dependent cleavage at physiological pH, which has not been reported for other bacterial LexA proteins, is possibly due to the absence of RecA interacting sites on Anabaena LexA protein, corresponding to the residues identified in E. coli LexA, and low cellular levels of RecA in Anabaena. Exposure to SOS-response inducing stresses, such as UV-B and mitomycin C neither affected the expression of LexA in Anabaena nor induced cleavage of LexA in either Anabaena 7120 or E. coli overexpressing Anabaena LexA protein. Though the LexA may be acting as a repressor by binding to the LexA box in the vicinity of the promoter region of specific gene, their derepression may not be via proteolytic cleavage during SOS-inducing stresses, unless the stress induces increase in cytoplasmic pH. This could account for the regulation of several carbon metabolism genes rather than DNA-repair genes under the regulation of LexA in cyanobacteria especially during high light induced oxidative stress.

Enhancement of MHC-I antigen presentation via architectural control of pH-responsive, endosomolytic polymer nanoparticles.

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell responses to protein-based vaccines.

Insights into the role of Asp79(2.50) in β2 adrenergic receptor activation from molecular dynamics simulations.

Achieving a molecular-level understanding of G-protein-coupled receptor (GPCR) activation has been a long-standing goal in biology and could be important for the development of novel drugs. Recent breakthroughs in structural biology have led to the determination of high-resolution crystal structures for the β2 adrenergic receptor (β2AR) in inactive and active states, which provided an unprecedented opportunity to understand receptor signaling at the atomic level. We used molecular dynamics (MD) simulations to explore the potential roles of ionizable residues in β2AR activation. One such residue is the strongly conserved Asp79(2.50), which is buried in a transmembrane cavity and becomes dehydrated upon β2AR activation. MD free energy calculations based on β2AR crystal structures suggested an increase in the population of the protonated state of Asp79(2.50) upon activation, which may contribute to the experimentally observed pH-dependent activation of this receptor. Analysis of MD simulations (in total > 100 μs) with two different protonation states further supported the conclusion that the protonated Asp79(2.50) shifts the conformation of the β2AR toward more active-like states. On the basis of our calculations and analysis of other GPCR crystal structures, we suggest that the protonation state of Asp(2.50) may act as a functionally important microswitch in the activation of the β2AR and other class A receptors.

Underlying thermodynamics of pH-dependent allostery.

Understanding the effects of coupling protein protonation and conformational states is critical to the development of drugs targeting pH sensors and to the rational engineering of pH switches. In this work, we address this issue by performing a comprehensive study of the pH-regulated switch from the closed to the open conformation in nitrophorin 4 (NP4) that determines its pH-dependent activity. Our calculations show that D30 is the only amino acid that has two significantly different pKas in the open and closed conformations, confirming its critical role in regulating pH-dependent behavior. In addition, we describe the free-energy landscape of the conformational change as a function of pH, obtaining accurate estimations of free-energy barriers and equilibrium constants using different methods. The underlying thermodynamic model of the switch workings suggests the possibility of tuning the observed pKa only through the conformational equilibria, keeping the same conformation-specific pKas, as evidenced by the proposed K125L mutant. Moreover, coupling between the protonation and conformational equilibria results in efficient regulation and pH-sensing around physiological pH values only for some combinations of protonation and conformational equilibrium constants, placing constraints on their possible values and leaving a narrow space for protein molecular evolution. The calculations and analysis presented here are of general applicability and provide a guide as to how more complex systems can be studied, offering insight into how pH-regulated allostery works of great value for designing drugs that target pH sensors and for rational engineering of pH switches beyond the common histidine trigger.

Determination of pH-dependent antioxidant activity of palm (Borassus flabellifer) polyphenol compounds by photoluminol and DPPH methods: a comparison of redox reaction sensitivity.

Palm juice (Borassus flabellifer) is one of the most common and cheap natural juices. Fermented palm juice contains various phytochemical compounds that exhibit antioxidant activity. In the present study, we examined the effects of pH on the production of phytochemicals and their antioxidant activity during the fermentation process. The concentration of total phenolics and flavonoid compounds of fermented palm juice and their antioxidant activity were investigated at various pH. The results showed that total phenolics concentration and antioxidant activity of palm wine and palm vinegar increase as pH increases: 3.5 < 4.5 < 5.5. Maximum flavonoid concentration was obtained at pH 6.5. Measurements of antioxidant activity by conventional DPPH method and Photochem antioxidant analyzer technique were highly correlated, with a corresponding R 2 value of 0.94.

Quinacrine inhibits Candida albicans growth and filamentation at neutral pH.

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 μg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment of C. albicans biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulated VMA3 mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-related Candida CR-BSI is warranted.

Structure analysis of disease-related proteins using vibrational spectroscopy

Analyses of the structure and properties of identified pathogenic proteins are important for elucidating the molecular basis of diseases and in drug discovery research. Vibrational spectroscopy has advantages over other techniques in terms of sensitivity of detection of structural changes. Spectral analysis, however, is complicated because the spectrum involves a substantial amount of information. This article includes examples of structural analysis of disease-related proteins using vibrational spectroscopy in combination with additional techniques that facilitate data acquisition and analysis. Residue-specific conformation analysis of an amyloid fibril was conducted using IR absorption spectroscopy in combination with (13)C-isotope labeling, linear dichroism measurement, and analysis of amide I band features. We reveal a pH-dependent property of the interacting segment of an amyloidogenic protein, β2-microglobulin, which causes dialysis-related amyloidosis. We also reveal the molecular mechanisms underlying pH-dependent sugar-binding activity of human galectin-1, which is involved in cell adhesion, using spectroscopic techniques including UV resonance Raman spectroscopy. The decreased activity at acidic pH was attributed to a conformational change in the sugar-binding pocket caused by protonation of His52 (pKa 6.3) and the cation-π interaction between Trp68 and the protonated His44 (pKa 5.7). In addition, we show that the peak positions of the Raman bands of the C4=C5 stretching mode at approximately 1600 cm(-1) and the Nπ-C2-Nτ bending mode at approximately 1405 cm(-1) serve as markers of the His side-chain structure. The Raman signal was enhanced 12 fold using a vertical flow apparatus.

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-14C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B′-MTs. It was concluded that CTgSs have strict substrate specificity. The Km values of CTgS1 and CTgS2 were 121 and 184μM with nicotinic acid as a substrate, and 68 and 120μM with S-adenosyl-l-methionine as a substrate, respectively.

Biochemical characterization of L-asparaginase in NaCl-tolerant Staphylococcus sp. OJ82 isolated from fermented seafood.

L-Asparaginase from gram-positive bacteria has been poorly explored. We conducted recombinant overexpression and purification of L-asparaginase from Staphylococcus sp. OJ82 (SoAsn) isolated from Korean fermented seafood to evaluate its biotechnological potential as an antileukemic agent. SoAsn was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of 37.5 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with asparaginases in gram-negative bacteria, size-exclusion chromatography determined SoAsn as a homodimer. Interestingly, the optimal temperature of SoAsn was 37°C and over 90% of activity was retained between 37°C and 50°C, and its thermal stability range was narrower than that of commercial E. coli L-asparaginase (EcAsn). Both SoAsn and EcAsn were active between pH 9 and 10, although their overall pH-dependent enzyme activities were slightly different. The Km value of SoAsn was 2.2 mM, which is higher than that of EcAsn. Among eight metals tested for enzyme activity, cobalt and magnesium greatly enhanced the SoAsn and EcAsn activity, respectively. Interestingly, SoAsn retained more than 60% of its activity under 2 M NaCl condition, but the activity of EcAsn was reduced to 48%. Overall, the biochemical characteristics of SoAsn were similar to those of EcAsn, but its kinetics, cofactor requirements, and NaCl tolerance differed from those of EcAsn.

PH dependent catalytic activities of platinum nanoparticles with respect to the decomposition of hydrogen peroxide and scavenging of superoxide and singlet oxygen.

Recently, platinum (Pt) nanoparticles (NPs) have received increasing attention in the field of catalysis and medicine due to their excellent catalytic activity. To rationally design Pt NPs for these applications, it is crucial to understand the mechanisms underlying their catalytic and biological activities. This article describes a systematic study on the Pt NP-catalyzed decomposition of hydrogen peroxide (H2O2) and scavenging of superoxide (O2˙(-)) and singlet oxygen ((1)O2) over a physiologically relevant pH range of 1.12-10.96. We demonstrated that the catalytic activities of Pt NPs can be modulated by the pH value of the environment. Our results suggest that Pt NPs possess peroxidase-like activity of decomposing H2O2 into ˙OH under acidic conditions, but catalase-like activity of producing H2O and O2 under neutral and alkaline conditions. In addition, Pt NPs exhibit significant superoxide dismutase-like activity of scavenging O2˙(-) under neutral conditions, but not under acidic conditions. The (1)O2 scavenging ability of Pt NPs increases with the increase in the pH of the environment. The study will provide useful guidance for designing Pt NPs with desired catalytic and biological properties.

Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0–9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.

Molecular dynamics simulations of histidine-containing cod antimicrobial peptide paralogs in self-assembled bilayers

Gaduscidin-1 and -2 (GAD-1 and GAD-2) are antimicrobial peptides (AMPs) that contain several histidine residues and are thus expected to exhibit pH-dependent activity. In order to help elucidate their mechanism of membrane disruption, we have performed molecular dynamics simulations with the peptides in both histidine-charged and histidine-neutral forms, along with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid molecules. The simulations employed GROMACS software and an OPLS-AA force field. Initially, the peptide and lipids were placed randomly in the simulation box and then were allowed to self-assemble. The results demonstrated a marked preference for the regions of the peptides that contain sequential pairs of histidine residues to associate closely with bilayer pores. This preference is observed even when the histidines are in their uncharged form. It appears that the relative compactness and rigidity of histidine pairs require the more aqueous and disordered environment of the pores to satisfy hydrophilic interactions. The final peptide structures exhibited a wide variety of structures and topologies, with the most helical structures positioning most parallel to the bilayer surface and the less ordered structures interacting more closely with the pore. Thus, the results give atomistic insight into those models of AMP mechanism that promote the importance of structural heterogeneity and imperfect amphipathicity to AMP activity and selectivity.

Species differences in bacterial NhaA Na+/H+ exchangers.

Bacteria have adapted their NhaA Na(+)/H(+) exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H(+) and Na(+). We show that H.pylori NhaA only has a small acidic shift of its pH-dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.

Characterization and expression of sweetfish (Pleco glossus altivelis) cathepsin D.

Cathepsin D (CTSD) is a lysosomal acidic endoproteinase that plays an important role in immune response. In this study, we obtained sweetfish (Plecoglossus altivelis) CTSD (PaCTSD) via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1 955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17 × 10³. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.

The potential roles of cell surface pHs in bioactive peptide activation.

Glycolytic metabolism of cells produces protons that are removed from the cytosol by transport proteins to create a pH difference between the adjacent bulk solution and the cell membrane surface. Therefore, tissue cells have distinct surface pHs because of varied glycocalyx and proton production capability. In this study, we proved the role of cell surface pH in peptide-cell interaction and peptide activation using lytic peptides with pH-dependent activity as probes. Properly, selected peptides could sense the specific pH zones on cells and thus demonstrated varied activity to tissue cells with different surface pHs. For a specific cell, the activity of pH-sensitive peptides changed accordingly as the cell surface pH was tuned up or down by proton channel regulators. Mechanistic studies revealed that cell surface pH directly affected peptide insertion into membranes by altering the secondary structure and aggregation status of membrane-bound pH-sensitive peptides. A pH-sensitive lytic peptide-designed based on the cell surface pH difference between a normal-cancer cell pair showed good selectivity to cancer cells. Therefore, cell surface pHs may present new opportunities to design therapeutic peptides with high cell specificity and selectivity.

Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis.

The novel species Halomonas zhaodongensis NEAU-ST10-25(T) recently identified by our group is a moderate halophile which can grow at the range of 0-2.5 M NaCl (optimum 0.5 M) and pH 6-12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25(T) in this study for Na(+)(Li(+))/H(+) antiporter genes by selection in Escherichia coli KNabc lacking three major Na(+)(Li(+))/H(+) antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.