Low Ionic Strength Conditions Research Articles

Modulating conformational stability of human serum albumin and implications for surface passivation applications

Human serum albumin (HSA) is one of the most abundant proteins in the human body and also used as a biomaterial coating. While there is significant interest in modifying the properties of solid supports to control HSA adsorption, there is an outstanding need to investigate whether modulating the conformational properties of HSA proteins can influence adsorption properties, especially in the context of surface passivation applications. Herein, we investigated how ionic strength – a key modulator of electrostatic forces influencing protein folding and protein-substrate interactions – can affect the conformational stability of HSA proteins and the resulting adsorption of HSA proteins onto silica surfaces for surface passivation applications. Thermal denaturation experiments identified that HSA is prone to unfolding and aggregation under physiologically relevant ionic strength conditions while HSA is more conformationally stable in low salt conditions. In line with these observations, we also determined that the adsorption of native HSA monomers onto silica surfaces was strongly influenced by electrostatic forces, with greater uptake in low ionic strength conditions. By contrast, the adsorption uptake of heat-denatured HSA was greatest in high ionic strength conditions and related to oligomer size. It was further revealed that native HSA is superior to heat-denatured HSA for passivating silica surfaces against serum biofouling. Taken together, our findings demonstrate that modulating the conformational stability of HSA proteins influences the adsorption pathway and passivation properties, offering fundamental insights that can lead to improved protein coatings.

Graphene oxide-facilitated uranium transport and release in saturated medium: Effect of ionic strength and medium structure

Natural subsurface environment is a complex heterogeneous system. To investigate the effect of ionic strength (IS) and heterogeneity on the transport and remobilization of graphene oxide (GO)-facilitated uranium (U(VI)) in saturated porous media, column experiments were performed by the injection of U(VI) alone and U(VI)+GO mixtures into homogeneous and heterogeneous porous media under low and high ionic strength (1 and 50 mM) conditions, and then the columns were successively flushed with background solution and DI water. Results showed that when U(VI) only was introduced into the columns, IS had little effect on the migration of U(VI) alone in both media and the presence of preferential flow in heterogeneous media slightly enhanced the mobility of U(VI). As U(VI)+GO mixtures were injected into the columns, GO showed strong mobility at low IS and high released peak at high IS. The appearance of GO significantly enhanced U(VI) transport in both media. Under low IS condition, the mobility of U(VI) was significantly enhanced at the injection phase, and the medium heterogeneity further promoted the amount of GO-sorbed U(VI) transport. At high IS, less GO-sorbed U(VI) was observed during injection phase, and a large amount of retained GO-sorbed U(VI) were released with GO remobilization during water flushing phase, and the release showed the longer-tailing phenomenon and the release amount was more pronounced in heterogeneous media. The findings in this study showed that the coupled effect of solution chemistry and media heterogeneity played important roles on GO-facilitated U(VI) transport and release in soil and groundwater system.

Biomolecular charges influence the response of surface plasmon resonance biosensors through electronic and ionic mechanisms

Surface plasmon resonance (SPR) biosensors have become an important label-free optical biomolecular sensing technology and a “gold standard” for retrieving information on the kinetics of biomolecular interactions. Even though biomolecules typically contain an abundance of easily ionizable chemical groups, there is a gap in understanding of whether (and how) the electrostatic charge of a biomolecular system influences the SPR biosensor response. In this work we show that negative static charge present in a biomolecular layer on the surface of an SPR sensor results in significant SPR spectral shifts, and we identify two major mechanisms responsible for such shifts: 1) the formation of an electrical double layer (ionic mechanism), and 2) changes in the electron density at the surface of a metal (electronic mechanism). We show that under low ionic strength conditions, the electronic mechanism is dominant and the SPR wavelength shift is linearly proportional to the surface concentration of biomolecular charges. At high ionic strength conditions, both electric and ionic mechanisms contribute to the SPR wavelength shift. Using the electronic mechanism, we estimated the pKa of surface-bound carboxylic groups and the relative concentration of the carboxyl-terminated alkanethiols in a binary self-assembled monolayer of alkanethiols. The reported sensitivity of SPR to surface charge is especially important in the context of biomolecular sensing. Moreover, it provides an avenue for the application of SPR sensors for fast, label-free determination of the net charge of a biomolecular coating, which is of interest in material science, surface chemistry, electrochemistry, and other fields.

Influence of Nano- and Microplastic Particles on the Transport and Deposition Behaviors of Bacteria in Quartz Sand

Plastic particles are widely present in the natural environment and are highly likely to interact with bacteria (the ubiquitous microbes in the natural environment), which might affect the transport and deposition of bacteria in porous media. In this study, the significance of plastic particles from nanoscale to micrometer-scale (0.02-2 μm) on the transport and deposition behaviors of bacteria ( Escherichia coli) in quartz sand was examined under environmentally relevant conditions in both NaCl and CaCl2 solutions at pH 6. The results showed that the presence of different-sized plastic particles did not affect bacterial transport behaviors at low ionic strength (10 mM NaCl and 1 mM CaCl2), whereas, at high ionic strength conditions (50 mM NaCl and 5 mM in CaCl2), plastic particles increased bacterial transport in quartz sand. At low ionic strength conditions, the mobility of both plastic particles and bacteria was high, which might drive the negligible effects of plastic particles on bacterial transport behaviors. The mechanisms driving the enhanced cell transport at high ionic strength were different for different-sized plastic particles. Specifically, for 0.02 μm nanoplastic particles, the adsorption of plastic particles onto cell surfaces and the repel effect induced by suspended plastic particles contributed to the increased cell transport. As for 0.2 μm microplastics (MPs), the suspended plastic particles induced repel effect contributed to the increased cell transport, whereas, for 2 μm MPs, the competition deposition sites by the plastic particles were the contributor to the increased cell transport.

Detection of unique Ebola virus oligonucleotides using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs

Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva. Equivalent levels of FRET occur using PMO probe pairs for single or double stranded oligonucleotide targets. High-affinity binding is retained under low-ionic strength conditions that disrupt oligonucleotide secondary structures (e.g., stem-loop structures), ensuring reliable target detection. Under these low-ionic strength conditions, rates of PMO probe binding to target oligonucleotides are increased 3-fold relative to conventional high-ionic strength conditions used for nucleic acid hybridization, with half-maximal binding occurring within 10 min. Our results indicate an ability to use PMO probe pairs to detect clinically relevant levels of EBOV and other oligonucleotide targets in complex biological samples without the need for nucleic acid amplification, and open the possibility of population screening that includes assays for the genomic integration of DNA based copies of viral RNA.

The Effect of Low Ionic Strength on Diffusion and Viscosity of Monoclonal Antibodies.

To determine the effect of solution conditions, especially low ionic strength, on the dynamics of molecular diffusion and protein-protein interactions in monoclonal antibody solutions. The interaction parameter, kD, was calculated from diffusion data obtained from dynamic light scattering (DLS) measurements performed using a Zetasizer. Theoretical considerations were utilized to evaluate the hard sphere and electrostatic contribution to molecular interactions. At low ionic strengths, repulsions were the dominant forces governing the behavior of both mAbs. As ionic strength increased, attractions contributed to the behavior of mAb1, while repulsions remained the dominant factor affecting mAb3 behavior. Repulsions alone were not sufficient to affect mAb3 viscosity in water, while the presence of repulsions as well as specific attractions was suggested to cause an increase in the viscosity of mAb1 in water compared to 15 mM ionic strength. Solution physical properties varied for the mAbs investigated. Our findings highlighted the importance of developing a fundamental understanding of interplay of forces governing solution properties of each individual mAb under low ionic strength conditions. Such understanding is critical in enabling successful development of self-buffered formulations.

Comparison of Types and Amounts of Nanoscale Heterogeneity on Bacteria Retention

Interaction energy calculations that assume smooth and chemically homogeneous surfaces are commonly conducted to explain bacteria retention on solid surfaces, but experiments frequently exhibit signification deviations from these predictions. A potential explanation for these inconsistencies is the ubiquitous presence of nanoscale roughness (NR) and chemical heterogeneity (CH) arising from spatial variability in charge (CH1), Hamaker constant (CH2), and contact angles (CH3) on these surfaces. We present a method to determine the mean interaction energy between a colloid and a solid-water-interface (SWI) when both surfaces contained binary NR and CH. This approach accounts for double layer, van der Waals, Lewis acid-base, and Born interactions. We investigate the influence of NR and CH parameters and solution ionic strength (IS) on interaction energy profiles between hydrophilic and hydrophobic bacteria and the SWI. Increases in CH1 and CH3 reduce the energy barrier and create deeper primary minima on net electrostatically unfavorable surfaces, whereas increasing CH2 diminishes the contribution of the van der Waals interaction in comparison to quartz and makes a more repulsive surface. However, these roles of CH are always greatest on smooth surfaces with larger fractions of CH. In general, increasing CH1 and CH3 have a larger influence on bacteria retention under lower IS conditions, whereas the influence of increasing CH2 is more apparent under higher IS conditions. However, interaction energy profiles are mainly dominated by small fractions of NR, which dramatically lower the energy barrier height and the depths of both the secondary and primary minima. This significantly increases the relative importance of primary to secondary minima interactions on net electrostatically unfavorable surfaces, especially for conditions that produce small energy barriers on smooth surfaces. Energy balance calculations indicate that this primary minimum is sometimes susceptible to diffusive removal depending on the NR and CH parameters.

High-Speed Lateral Flow Strategy for a Fast Biosensing with an Improved Selectivity and Binding Affinity.

We report a high-speed lateral flow strategy for a fast biosensing with an improved selectivity and binding affinity even under harsh conditions. In this strategy, biosensors were fixed at a location away from the center of a round shape disk, and the disk was rotated to create the lateral flow of a target solution on the biosensors during the sensing measurements. Experimental results using the strategy showed high reaction speeds, high binding affinity, and low nonspecific adsorptions of target molecules to biosensors. Furthermore, binding affinity between target molecules and sensing molecules was enhanced even in harsh conditions such as low pH and low ionic strength conditions. These results show that the strategy can improve the performance of conventional biosensors by generating high-speed lateral flows on a biosensor surface. Therefore, our strategy can be utilized as a simple but powerful tool for versatile bio and medical applications.

Adsorption Properties of Microwave Modified Diatomite on Heavy Metals in Landfill Leachate

Adsorption properties of diatomite modified by microwave on lead, chromium and cadmium in landfill leachate under different conditions were studied with diatomite as adsorbent, and the best microwave modification conditions were determined in this study. Meanwhile, the adsorption and removal effects of diatomite on Pb, Cr and Cd in landfill leachate under different adsorption conditions were investigated and which including adsorbent dosage, adsorption time, temperature, pH and the ion strength. The results showed that the best adsorption removal efficient of heavy metal ion in landfill leachate can be obtained under the adsorption conditions of low ionic strength, alkaline leachate, with adsorption temperature of 25 °C, the adsorption time of 40 min, and the modified diatomite dosing quantity of 5∼10g/L.

Ion concentration in micro and nanoscale electrospray emitters.

Solution-phase ion transport during electrospray has been characterized for nanopipettes, or glass capillaries pulled to nanoscale tip dimensions, and micron-sized electrospray ionization emitters. Direct visualization of charged fluorophores during the electrospray process is used to evaluate impacts of emitter size, ionic strength, analyte size, and pressure-driven flow on heterogeneous ion transport during electrospray. Mass spectrometric measurements of positively- and negatively-charged proteins were taken for micron-sized and nanopipette emitters under low ionic strength conditions to further illustrate a discrepancy in solution-driven transport of charged analytes. A fundamental understanding of analyte electromigration during electrospray, which is not always considered, is expected to provide control over selective analyte depletion and enrichment, and can be harnessed for sample cleanup. Graphical abstract Fluorescence micrographs of ion migration in nanoscale pipettes while solution is electrosprayed.

Improving accuracy of electrochemical capacitance and solvation energetics in first-principles calculations

Reliable first-principles calculations of electrochemical processes require accurate prediction of the interfacial capacitance, a challenge for current computationally efficient continuum solvation methodologies. We develop a model for the double layer of a metallic electrode that reproduces the features of the experimental capacitance of Ag(100) in a non-adsorbing, aqueous electrolyte, including a broad hump in the capacitance near the potential of zero charge and a dip in the capacitance under conditions of low ionic strength. Using this model, we identify the necessary characteristics of a solvation model suitable for first-principles electrochemistry of metal surfaces in non-adsorbing, aqueous electrolytes: dielectric and ionic nonlinearity, and a dielectric-only region at the interface. The dielectric nonlinearity, caused by the saturation of dipole rotational response in water, creates the capacitance hump, while ionic nonlinearity, caused by the compactness of the diffuse layer, generates the capacitance dip seen at low ionic strength. We show that none of the previously developed solvation models simultaneously meet all these criteria. We design the nonlinear electrochemical soft-sphere solvation model which both captures the capacitance features observed experimentally and serves as a general-purpose continuum solvation model.

The Mixing Counterion Effect on DNA Compaction and Charge Neutralization at Low Ionic Strength.

DNA compaction and charge neutralization in a mixing counterion solution involves competitive and cooperative electrostatic binding, and sometimes counterion complexation. At normal ionic strength, it has been found that the charge neutralization of DNA by the multivalent counterion is suppressed when being added extra mono- and di-valent counterions. Here, we explore the effect mixing counterion on DNA compaction and charge neutralization under the condition of low ionic strength. Being quite different from normal ionic strength, the electrophoretic mobility of DNA in multivalent counterion solution (octalysine, spermine) increases the presence of mono- and di-valent cations, such as sodium and magnesium ions. It means that the charge neutralization of DNA by the multivalent counterion is promoted rather than suppressed when introducing extra mono- and di-valent counterions into solution. This conclusion is also supported by the measurement of condensing and unraveling forces of DNA condensates under the same condition by single molecular magnetic tweezers. This mixing effect can be attributed to the cooperative electrostatic binding of counterions to DNA when the concentration of counterions in solution is below a critical concentration.

Marmoset cytochrome P450 2B6, a propofol hydroxylase expressed in liver

The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available.Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents.Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys.Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low Km value was relatively comparable to that for marmoset livers.These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

Electrostatic confinement and manipulation of DNA molecules for genome analysis

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte "issues" as exploitable advantages by our invention and characterization of the "molecular gate," which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an "electrostatic bottle." This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasmaflorum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

Determination of electroosmotic and electrophoretic mobility of DNA and dyes in low ionic strength solutions.

Nanocoding, a genome analysis platform, relies on very low ionic strength conditions to elongate DNA molecules up to 1.06 (fully stretched DNA=1). Understanding how electroosmotic and electrophoretic forces vary, as ionic strength decreases, will enable better Nanocoding devices, or other genome analysis platforms, to be developed. Using gel electrophoresis to determine overall mobility (includes contributions from electrophoretic and electroosmotic forces) in different ionic strength conditions, linear DNA molecules (pUC19 (2.7 kb), pBR322 (4.4 kb), ΦX174 (5.4 kb), and PSNAPf-H2B (6.2 kb)) were analyzed in varying gel concentrations (1.50, 1.25, 1.00, 0.75, and 0.50%). Additionally, buffer concentration (Tris-EDTA, TE) was varied to determine free solution mobility at different ionic strength solutions. As ionic strength decreased from 13.8 to 7.3mM, overall mobility increased. As TE buffer decreased (< 7.3mM), overall mobility drastically decreased as ionic strength decreased. Rhodamine B dye was utilized to determine the electroosmotic mobility. As the ionic strength decreased, electroosmotic mobility increased. The experimental electrophoretic mobility was compared to theoretical considerations for electrophoretic mobility (Pitts and Debye-Hückel-Onsager). Electroosmotic forces decreased the overall mobility of DNA molecules and bromophenol blue migration in a gel matrix as ionic strength decreased.

Parallel-plate fracture transport experiments of nanoparticulate illite in the ultra-trace concentration range investigated by Laser-Induced Breakdown Detection (LIBD)

This study investigates the appropriateness of monodisperse carboxylated polystyrene spheres as clay colloid analogues. Colloid transport experiments using Na-illite are conducted within a parallel-plate fracture flow cell with an aperture of 0.75mm at pH 5 under low ionic strength (1mM NaCl) and laminar flow (7mL/h) conditions. The effects of collector surface material (Grimsel granodiorite or acrylic glass), fracture orientation and residence time on colloid retention are examined. In order to avoid full surface coverage effects, ultra-trace (30ppb) Na-illite colloid concentrations are used. Laser-induced breakdown detection is applied in order to detect and quantify the Na-illite colloids. The results are directly compared to the experiments of Stoll, et al. [1] who investigated the transport and surface interaction of carboxylated polystyrene spheres (25 and 1000nm diameter) using the same experimental setup and conditions. It is concluded that polystyrene spheres are no ideal analogue to predict the mobility and transport behavior of a polydisperse clay colloid suspension. Despite the higher solid density, the Na-illite colloids show higher mobility compared to the polystyrene spheres without significant impact of surface roughness or gravity. The platelet shape, the non-uniform surface charge distribution and slower sedimentation may account for the observed differences.

Surface-anchored counterions on weak chiral anion-exchangers accelerate separations and improve their compatibility for mass-spectrometry-hyphenation

In the present work we propose new variants of chiral stationary phases (CSP) with tert-butylcarbamoylquinine (tBuCQN) as chiral selector molecule. Four tBuCQN-CSPs with distinct bonding chemistries are compared in terms of their pH-dependent surface charge by ζ-potential determinations, by achiral and chiral liquid chromatographic tests and LC–ESI–MS hyphenation. In one embodiment tBuCQN was immobilized on 3-mercaptopropylmethylsilyl-modified silica by thiol-ene click reaction (brush type CSP with selector coverage of 0.38mmol/g). In another embodiment, poly-(3-mercaptopropyl)-methylsiloxane was coated onto vinylized silica particles in presence of tBuCQN and radical initiator. The tBuCQN selector was then immobilized onto the polysiloxane film which in turn was crosslinked to the vinyl-surface in a simultaneous double click reaction leading to a CSP with enhanced stability due to multiple linkages (0.29mmol/g tBuCQN). Aliquots of each of the two CSPs were further modified by oxidation of free residual thiol groups to sulfonic acid functionalities to obtain strongly acidic endcapping groups which act as immobilized counterions of the chiral WAX CSPs (0.2mmol/g sulfonic acid co-ligands for brush type CSP). This caused secondary repulsive interactions, hence balanced interactions of the target analytes (chiral acids) at the WAX site and decreased non-specific interactions. Furthermore, this rendered possible the use of milder elution conditions, i.e. lower ionic strength, for acidic compounds. Separation performance was maintained and slightly improved, respectively, when using polar organic or reversed-phase type elution mode in chiral separations which were significantly accelerated (isoeluotropic conditions could be achieved with ca. factor 40 lower counterion concentration in the mobile phase). Thus, LC–ESI–MS enantiomer separations could be readily performed at very low ionic strength conditions (10mM acetate) which is favorable due to less ion suppression. In addition to this the newly developed stationary phases showed complementary retention profiles in RP- and HILIC-mode which make these type of stationary phases also promising tools for achiral applications in pharmaceutical analysis, especially as orthogonal separation principle e.g. in 2D-LC and impurity profiling.

Molecular Recognition in Mitochondrial Cytochromes P450 That Catalyze the Terminal Steps of Corticosteroid Biosynthesis

The mitochondrial cytochromes P450 11B1 and P450 11B2 are responsible for the final stages of cortisol and aldosterone synthesis, respectively. Dysregulation of both enzymes has been implicated in secondary forms of hypertension. Molecular recognition of the cytochromes P450 with their corresponding redox partner is a key step in the catalytic cycle, yet the precise nature of the interaction of P450 11B1 or P450 11B2 with their proximal partner, adrenodoxin (Adx), is still unknown. Here, we obtained P450 11B1·Adx2 and P450 11B2·Adx2 complexes using the zero-length cross-linker ethyl-3-[3-(dimethylamino)propyl]carbodiimide, which formed best under low-ionic strength conditions. R-to-K mutations were introduced into the P450s at residues predicted to form salt bridges with Adx and allow cross-linking with the carbodiimide reagent. Mass spectrometric analysis of the chymotrypsin-digested ternary complexes identified seven cross-linked peptide pairs. Consistent with the electrostatic interaction of K370 in P450 11B1-WT and K366 in P450 11B2-R366K with D79 of Adx, Adx mutation L80K abolished complex formation. Using these sites of interaction as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the P450 11B1·Adx2 complex. The appositional surfaces include R/K366, K370, and K357 of P450 11B1, which interact with D79, D76, and D113 (second molecule) of Adx, respectively. Similar to P450 11B1, P450 11B2 also forms a complex with the Adx dimer via three lysine residues. We describe similarities and differences in our models of the P450 11B1·Adx2 and P450 11B2·Adx2 complexes with the structure of the P450 11A1-Adx fusion protein.

Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (Kd ≈ 10-8 to 10-9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd ≈ 10-7 M), but those retaining exon 10 (ABR+10) did so only weakly (Kd ≈ 10-5 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg2+ ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.

Pyrophosphate hydrolysis in the extremely halophilic archaeon Haloarcula japonica is catalyzed by a single enzyme with a broad ionic strength range.

The soluble protein fraction of the extremely halophilic archaeon Haloarcula japonica exhibits substantial inorganic pyrophosphate (PPi) hydrolysis activity in the presence of 2-4M NaCl (Wakai et al, J Biol Chem 288:29247-29251, 2013), which provides high ionic strength (2-4). In this study, much higher PPi hydrolysis activity was unexpectedly detected, even with 0M NaCl in the presence of 100-200mM MgSO4, providing a much lower ionic strength of 0.4-0.8, in the same protein fraction. Na+ and Mg2+ ions were required for activity under high and low ionic strength conditions, respectively. A recombinant H. japonica pyrophosphatase (HjPPase) exhibited PPi hydrolysis activity with the same broad ionic strength range, indicating that the activity associated with such a broad ionic strength range could be attributed to a single enzyme. Thus, we concluded that the broad ionic strength range of HjPPase may contribute to adaptation for both Na+ and Mg2+ which are abundant but variable in the unstable living environments of H. japonica.