Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Preparation of capacitor’s electrode from sunflower seed shell

Series of nanoporous carbons are prepared from sunflower seed shell (SSS) by two different strategies and used as electrode material for electrochemical double-layer capacitor (EDLC). The surface area and pore-structure of the nanoporous carbons are characterized intensively using N₂ adsorption technique. The results show that the pore-structure of the carbons is closely related to activation temperature and dosage of KOH. Electrochemical measurements show that the carbons made by impregnation-activation process have better capacitive behavior and higher capacitance retention ratio at high drain current than the carbons made by carbonization-activation process, which is due to that there are abundant macroscopic pores and less interior micropore surface in the texture of the former. More importantly, the capacitive performances of these carbons are much better than ordered mesoporous carbons and commercial wood-based active carbon, thus highlighting the success of preparing high performance electrode material for EDLC from SSS.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

High frequency residues: A new set of signals for detectability studies of an X-ray imaging system

A new set of signals for studying detectability of an X-ray imaging system is presented. The results obtained with these signals are intended to complement the NEQ results.The signals are generated from line spread profiles by progressively removing their lower frequency components and the resulting high frequency residues (HFRs) form the set of signals to be used in detectability studies. Detectability indexes for these HFRs are obtained using a non-prewhitening (NPW) observer and a series of edge images are used to obtain the HFRs, the covariance matrices required by the NPW model and the MTF and NPS used in NEQ calculations. The template used in the model is obtained by simulating the processes of blurring and sampling of the edge images. Comparison between detectability indexes for the HFRs and NEQ are carried out for different acquisition techniques using different beam qualities and doses.The relative sensitivity shown by detectability indexes using HFRs is higher than that of NEQ, especially at lower doses. Also, the different observers produce different results at high doses: while the ideal Bayesian observer used by NEQ distinguishes between beam qualities, the NPW used with the HFRs produces no differences between them.Delta functions used in HFR are the opposite of complex exponential functions in terms of their support in the spatial and frequency domains. Since NEQ can be interpreted as detectability of these complex exponential functions, detectability of HFRs is presented as a natural complement to NEQ in the performance assessment of an imaging system.     

The effect of dielectric polarization of the electrode on anomalous temperature effects in the electrical double layer

Monte Carlo simulations and a modified Poisson–Boltzmann (MPB) theory are used to investigate the temperature dependence of the capacitance (around the potential of zero charge) of an electric double layer in the presence of surface polarization due to a dielectric boundary. Within the context of the restricted primitive model planar double layer, whose solvent dielectric constant is ε₂, the cases when the electrode is an insulator (ε₁ = 1), when the electrode and the electrolyte have the same permittivity (ε₁ = ε₂, no polarization), and when the electrode is a conductor (ε₁ → ∞) are studied for the case where the electrolyte concentration is 0.1 M. The simulations reveal a capacitance anomaly, that is, a positive temperature dependence of the capacitance at low temperatures for the former two situations. The MPB theory also shows this effect for these two situations and is in qualitative or better agreement with the simulation data. In these two cases, both the simulations and theory show a dramatic increase of the diffuse layer potential in the temperature regime where capacitance anomaly occurs. However, in the latter situation, where the electrode is metallic, the capacitance always has a negative temperature derivative for the MPB theory and probably also for the simulation data.     

Dot-immunobinding assay in the detection of IgG antibodies against farmer's lung antigens

Circulating antibodies against Faenia rectivirgula, Thermoactinomyces candidus, T. vulgaris and Aspergillus fumigatus were studied in the sera of 14 clinically proven farmer's lung patients and 10 normal controls using three immunological methods. These methods were agar gel double diffusion (DD), biotin-avidin-linked immunosorbent assay (BALISA) and dot-immunobinding assay (DIBA). Agar gel diffusion, the least sensitive of the three methods, failed to detect antibodies in some of the patients, while BALISA detected antibodies even in the normal controls. However, the sensitivity of dot-immunobinding assay was in between DD and BALISA while the specificity was comparable to DD to all the antibodies except against A. fumigatus antigens. Dot-immunobinding assay gave faster results than DD and the blots can be stored as record for longer periods of time without fading.     

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.     

Calcium at the surface of cardiac plasma membrane vesicles: Cation binding,surface charge screening,and Na−Ca exchange

Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m ^–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca].