Reservoir Buffer Research Articles

Can reservoir and groundwater storage buffer the impacts of climate-induced declines in snowpack storage for agriculture and the environment? Insights from the Walker River Basin

Climate-induced declines in snowpack storage have profound consequences for snowmelt-dependent river basins globally, including those in the western United States. These basins face the risk of economic losses in agriculture and environmental damage due to disrupted instream flows. To mitigate these potential losses, two strategies are commonly employed: enhancing built reservoir storage capacity and increasing groundwater use during the irrigation season. However, implementing these strategies carries the risk of exacerbating instream flow disruptions and compromising the sustainability of aquifers. This article develops a hydro-economic optimization model of Nevada's Walker River Basin (WRB) and uses it to examine the impact of storage capacity and groundwater management on mitigating economic losses in agriculture caused by altered snowmelt-driven inflows. The model accounts for environmental constraints on instream flows that limit water availability for agricultural use. Results show that the WRB is projected to experience a decline in irrigated farm acreage and agricultural output due to reduced snowpack storage. The findings also indicate diminishing returns on reservoir capacity investments, while highlighting the increased value of these investments when groundwater pumping is constrained due to aquifer sustainability concerns. Results show that upgrading existing reservoir capacity by not more than 1.5% in a free water leasing market could protect upwards of 94% and 83% of baseline agricultural income when the basin faces future climate-induced early season snowmelt-driven flow timing, and reduced streamflow, respectively, while managing the basin for hydrologic and environmental constraints.

Petrophysical Properties of Mauddud Formation in Selected Wells in Al-Ahdab Oil Field, Middle Iraq

This paper aims to calculate the petrophysical properties in the Al-Ahdab field in the middle of Iraq within the Mauddud Formation. This study was based on the information available from well logs. The interactive petrophysical software IP (V4.5) was used to calculate the porosity, hydrocarbon saturation and shale volume, divide the formation into reservoir units and buffer units, and evaluate these units in each well. The Mauddud was divided into five units, two of them were considered good reservoirs having good petrophysical properties (high porosity, Low water saturation, and low shale volume). The other three are not reservoirs because of poor petrophysical properties.

The prospect of kemiri sunan (Reutalis trisperma B. airy shaw) development as a source of bio-oil from inedible crops

Kemiri Sunan [Reutealis trisperma (Blanco) Airy Shaw] is a crop oil-producing plant with great potential as a source of raw materials for the oleochemical, biofuels, and other derivate product industries. Here, we focus on the prospect and potential of the kemiri sunan plant as a potential source of biofuel from inedible crops. The main potential lies in the fruit, which a dry seed production rate of 10-15 tons/ha/year or equivalent to about 8 tonnes of crude oil/ha/yr or about 7 tonnes of biodiesel/ha/yr. This uniqueness can be used as a source of raw materials for biofuel and other chemical industries that do not compete with crop oils for food. However, its current development in Indonesia is still constrained by the availability of land. The alternative that can be taken is to utilize unproductive land, including sub-optimal land, ex-mining land, and other degraded lands. Constraints of limited land for the development of kemiri sunan can be overcome by utilizing less productive lands, including ex-mining land, dry climate dry land, and reservoir buffer land. The condition of the land is sufficient to support the growth and development of the kemiri sunan plant which is quite good. The plant's character is in the form of a tree with a shady leaf crown and a deep root system that makes this plant very useful as a conservation plant on these lands.

Improving the accuracy of nonpoint-source pollution estimates in inland waters with coupled satellite-UAV data

Quantitatively and accurately analyzing nonpoint-source (NPS) pollution is essential for efficiently preventing the input of NPS loads into inland waters. However, the accuracy of previous NPS pollution models is limited by the accuracy of ground parameter data. In addition, there are few effective methods that thoroughly verify modeling results at large scales. This paper presents a framework for accurate NPS pollution estimation by coupling satellite and unmanned aerial vehicle (UAV) monitoring data, and the results are verified by both field sampling and a newly developed inlet NPS pollution “observation” simulation method. Fractional vegetation coverage (FVC) data obtained by satellite were used to improve the accuracy of the runoff module of the framework. Satellite and UAV data were coupled to acquire livestock data, determine inlets, and identify reservoir buffer zones and vegetation types. These new data were then used to improve the accuracy of the livestock and runoff modules in the framework. The results show that the estimation accuracy of total nitrogen, total phosphorus, ammonia nitrogen, and chemical oxygen demand with FVC were improved by 39.96%, 69.29%, 54.05% and 47.22% (in relative error), respectively. The high-resolution livestock data acquisition improved the estimation accuracy of the NPS pollution load by 7–53%. The high-resolution inlet extraction improved the accuracy by 3–24%. The high-resolution buffer zone identification improved the accuracy with the estimated NPS pollutant concentration into reservoir decreasing by 60–99%. Finally, the high-resolution vegetation type identification improved the accuracy by 10–72%. The framework performs satisfactorily, which was verified based on the simulated NPS observations with an average relative error of 11.54–24.31%. We found that the FVC, livestock number, and inlet number are key parameters for NPS pollution modeling; the introduction of monthly variation in the FVC makes the modeled NPS pollution load much higher in areas with mature complex forested ecosystems or densely distributed vegetation but much lower in areas with sparsely distributed vegetation. The above methods provide a scientific reference for high-efficiency NPS pollution prevention in inland waters, laying a solid basis for decision-making regarding water quality management in data-scarce regions around the world.

The Relationship between Soil Particle Size Fractions, Associated Carbon Distribution and Physicochemical Properties of Historical Land-Use Types in Newly Formed Reservoir Buffer Strips

Water impoundment reshapes the ecological environment around the bank-line of newly built reservoirs. Therefore, reservoir buffer strips play a disproportionately large role in the maintenance of ecosystem functions and environmental benefits during the early stage of reservoir formation. However, there are gaps in the research on soil particle-size-associated carbon distribution characteristics within different historical land-use types in newly formed reservoir buffer strips. In this study, we focused on soil particle size fractions, aggregate stability, and particle-size-associated carbon distribution characteristics of different historical land-use types of reservoir buffer strips at distance scale (i.e., different distance from the water) after reservoir impoundment in the Chushandian Reservoir, China, and explored the relationship between them. The results showed that the soil texture of abandoned cropland and grassland are classified as silt loam and woodland are classified as sandy loam; different historical land-use types in newly formed reservoir buffer strips showed significant differences in soil aggregate stability after reservoir impoundment; a distance scale was used to measure these differences, which were mainly due to the dry-wet cycles and water submerged condition caused by the buffers’ different distances from water. The newly formed reservoir buffer strips underwent corresponding changes in the particle-size-associated carbon distribution characteristics after reservoir impoundment, mainly due to the turnover property of different soil particles combined with organic carbon. Reservoir impoundment accelerates the turnover of silt particle and associated nutrients in soils of historical land-use types in newly formed reservoir buffer strips; turnover may be mediated mainly by microbial biomass.

Electrophoretic Separation of Very Large Molecular Weight Proteins in SDS Agarose.

Very large proteins (subunit sizes, >200kDa) are difficult to electrophoretically separate on polyacrylamide gels. A SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. Proteins with molecular masses between 200 and 4000kDa can be clearly separated. Inclusion of a reducing agent in the upper reservoir buffer and use of a large pore-sized agarose have been found to be key technical procedures for obtaining optimum protein migration and resolution.

The adsorption/desorption of phosphorus in freshwater sediments from buffer zones: the effects of sediment concentration and pH.

Riparian buffer zones adjacent to reservoirs and lakes protect aquatic ecosystems from polluted surface runoff. Sediments, collected from the buffer zones of Danjiangkou Reservoir (SR) and Honghu Lake (SL) in an ecologically fragile region in central China, were evaluated to reveal their phosphorus-adsorbing/desorbing properties and storage capacities. A nonlinear regression method was used to fit the pseudo-second-order kinetic and the modified crossover-type Langmuir isotherm models to the experimental data. It is shown that the adsorption of phosphorus onto the studied sediments followed the pseudo-second-order kinetic expression. The modified crossover-type Langmuir isotherm model was found to be a suitable method for describing adsorption/desorption processes in the experimental sediments. The maximum adsorption capacities (Q m), partitioning coefficients (K p), native adsorbed exchangeable phosphorus (NAP), and equilibrium phosphorus concentration (EPC0) were subsequently obtained for the experimental sediments. The effects of sediment concentration and pH were also investigated by batch experiments and Fourier transformation infrared and scanning electron microscopy analyses. The adsorption/desorption characteristics of different phosphate species on the sediments from reservoir and lake buffer zones were identified.

Method for resolution and western blotting of very large proteins using agarose electrophoresis.

Proteins larger than 200 kDa are difficult to separate electrophoretically using polyacrylamide gels, and their transfer during western blotting is typically incomplete. A vertical SDS agarose gel system was developed that has vastly improved resolving power for very large proteins. Complete transfer of proteins as large as titin (Mr 3,000-3,700 kDa) onto blots can be achieved. The addition of a sulfhydryl reducing agent in the upper reservoir buffer and transfer buffer markedly improves the blotting of large proteins.

Crystal structures of LsrR complexed with p-AI-2 reveal distinct mechanisms

Quorum sensing (QS) is a cell-to-cell communication system and responsible for a variety of bacterial phenotypes including virulence and biofilm formation. QS is mediated by small molecules, autoinducers (AIs), including AI-2 that is secreted by both Gram (+/–) microbes. LsrR is a key transcriptional regulator that governs the varied downstream processes by perceiving AI-2 signal, but its activation via autoinducer-binding remains poorly understood [1]. The ligand-free crystals of LsrR and complex crystals of LsrR and C-LsrR with 5 mM R5P were grown with reservoir buffer. Complex crystal of C-LsrR/D5P and C-LsrR/D8P were obtainded by soaking the native crystals in the same crystallization buffer (pH 6.5, 0.1 M bis-tris, 9.1% PEG-3350, 10 mM barium chloride dehydrate, 10 mM R5P) containing 0.15mM D5P and 2.0 mM D8P. These crystals were determined its 3-demensional (3D) structure at 3.2 Å ~ 1.9 Å resolution after SAD phasing. The ligand-binding affinities for LsrR protein were measured using fluorescence spectrophotometer and Isothermal titration calorimetry (ITC) while increasing the ligand concentrations. Detailed regulatory mechanism of LsrR from the crystal structures in complexes with the native signal (phospho-AI-2, D5P) and two quorum quenching antagonists (ribose-5-phosphate, R5P; phosphoisobutyl-AI-2, D8P). The bound D5P and D8P molecules are not the diketone forms but rather hydrated, and the hydrated moiety forms important H-bonds with the carboxylate of D243. The D5P-binding flipped out F124 of the binding pocket, and resulted in the disruption of the dimeric interface-1 by unfolding the α7 segment. However, the same movement of F124 by the D8P'-binding did not cause the unfolding of the α7 segment. Although the LsrR-binding affinity of R5P (Kd, ~1 mM) is much lower than those of D5P and D8P (~2.0 and ~0.5 μM), the α-anomeric R5P molecule fits into the binding pocket without any structural perturbation, and thus stabilizes the LsrR tetramer. The binding of D5P, not D8P and R5P, disrupted the tetrameric structure and thus is able to activate LsrR. The detailed structural and mechanistic insights from this study could be useful for facilitating design of new anti-virulence and anti-biofilm agents based on LsrR.

Identifying the Relationships between Water Quality and Land Cover Changes in the Tseng-Wen Reservoir Watershed of Taiwan

The effects on water quality of land use and land cover changes, which are associated with human activities and natural factors, are poorly identified. Fine resolution satellite imagery provides opportunities for land cover monitoring and assessment. The multiple satellite images after typhoon events collected from 2001 to 2010 covering land areas and land cover conditions are evaluated by the Normalized Difference Vegetation Index (NDVI). The relationship between land cover and observed water quality, such as suspended solids (SS) and nitrate-nitrogens (NO3-N), are explored in the study area. Results show that the long-term variations in water quality are explained by NDVI data in the reservoir buffer zones. Suspended solid and nitrate concentrations are related to average NDVI values on multiple spatial scales. Annual NO3-N concentrations are positively correlated with an average NDVI with a 1 km reservoir buffer area, and the SS after typhoon events associated with landslides are negatively correlated with the average NDVI in the entire watershed. This study provides an approach for assessing the influences of land cover on variations in water quality.

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates

We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.

Crystal Structure of Yeast Rpn14, a Chaperone of the 19 S Regulatory Particle of the Proteasome

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

Assessment of the long-term safety of radioactive waste disposal: 2. Isotopic study of water exchange in a multilayer system

As a continuation of previous studies of 3H, δ2H, δ18O, Ar, 40Ar/36Ar, and 234U/238U isotope systems, data of uranium-helium dating of groundwater at the radioactive waste repository of Siberian Chemical Works are quantitatively interpreted. The interpretation is based on analytical models taking into account the inflow of deep-earth helium and the diffusion-advection exchange between layers. Estimated flow velocities in reservoir beds and buffer horizons are in agreement with previous hydrodynamic estimates. The obtained velocities of horizontal mass transfer were used to evaluate the age of groundwater in sampling points. Water age varies from 7 to 14 (with a mean of ∼9) in the lower (II and III) aquifers, into which the radioactive wastes are pumped, from 2 to 4 in buffer horizon IV, and from 1 to 2 thousand years in the upper V horizon.

Up-regulation of Nicotinic Receptors by Nicotine Varies with Receptor Subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.

The Structure of a Full-length Response Regulator from Mycobacterium tuberculosis in a Stabilized Three-dimensional Domain-swapped, Activated State

The full-length, two-domain response regulator RegX3 from Mycobacterium tuberculosis is a dimer stabilized by three-dimensional domain swapping. Dimerization is known to occur in the OmpR/PhoB subfamily of response regulators upon activation but has previously only been structurally characterized for isolated receiver domains. The RegX3 dimer has a bipartite intermolecular interface, which buries 2357 A(2) per monomer. The two parts of the interface are between the two receiver domains (dimerization interface) and between a composite receiver domain and the effector domain of the second molecule (interdomain interface). The structure provides support for the importance of threonine and tyrosine residues in the signal transduction mechanism. These residues occur in an active-like conformation stabilized by lanthanum ions. In solution, RegX3 exists as both a monomer and a dimer in a concentration-dependent equilibrium. The dimer in solution differs from the active form observed in the crystal, resembling instead the model of the inactive full-length response regulator PhoB.

Structural Basis of Na+ Activation Mimicry in Murine Thrombin

Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na+-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na+ binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na+ pore in the murine enzyme. These findings demonstrate that Na+ activation in thrombin is linked to the architecture of the Na+ pore. The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.

Structural Basis for the Conformational Integrity of the Arabidopsis thaliana HY5 Leucine Zipper Homodimer

The leucine zipper (LZ) domain of the HY5 transcription factor from Arabidopsis thaliana has unique primary structural properties, including major occupation by the Leu residues as well as two buried polar residues in the a positions and a localized distribution of charged and polar residues in the first three heptad repeats. In this study, we solved the crystal structure of the HY5 LZ domain and show that the peculiarities in the primary sequence yield unusual structural characteristics. For example, the HY5 LZ domain exhibits a bipartite charge distribution characterized by a highly negative electrostatic surface potential in its N-terminal half and a nearly neutral potential in its C-terminal half. The LZ N-terminal region also contains two consecutive putative trigger sites for dimerization of the coiled coils. In addition, two buried asparagines at a positions 19 and 33 in the HY5 LZ domain display distinct modes of polar interaction. Whereas Asn(19) shows a conformational flip-flop, Asn(33) is engaged in a permanent hydrogen bond network. CD spectropolarimetry and analytical ultracentrifugation experiments performed with versions of the HY5 LZ domain containing mutations in the a positions yielded further evidence that position a amino acid residues are crucial for achieving an oligomeric state and maintaining stability. However, a low correlation between position a amino acid preference, core packing geometry, and rotamer conformations suggests that the oligomeric state of the LZ domain is not governed entirely by known structural properties. Taken together, our results suggest structural factors conferring conformational integrity of the HY5 LZ homodimer that are more complicated than proposed previously.

Sunset Yellow: A Food Color for Protein Staining with SDS-PAGE

This study shows that Sunset Yellow FCF is a fast-reacting dye for food proteins and may be used as a fast staining agent for all types of resolved proteins with PAGE, which is important because Coomassie Brilliant Blue R 250 and Amido Black 10 B take at least 24 hr for visualization of proteins bands with PAGE. Sunset Yellow FCF is highly sensitive, rapid, lasting, and provides reproducible results with a variety of proteins. It produces sharp, nondiffuseable, clear, distinct, and stable bands on gel. It requires just 1 hr for staining and destaining to visualize the known proteins separated on the acrylamide gels. intestinal anaerobes (5). The dye may be purified by a chemometric method (6) and, even in its crude form, is equally effective for protein staining on acrylamide and for use after blotting (7). Apart from Coomassie Brilliant Blue, the other standard protocols of dyes include silver and gold staining (8–10) and Amido Black 10 B (11); iodine is less commonly used (12). Although the spectrophotometric determination and estimation of Sunset Yellow FCF at 485 nm is common, capillary electrophoresis has recently been used as a sophisticated technique to estimate colors in foods, including this dye in beverages (13,14). The nanomaterials in foods, such as micronutrients and synthetic colors like Sunset Yellow FCF, may be analyzed by the above separation technique. The dyebinding assays are widely used in protein analysis. Our data show that Sunset Yellow FCF is a sensitive, reliable, and competent dye for binding proteins to make them visible with PAGE. MATERIALS AND METHODS Chemicals N,N′-methylene-bis-acrylamide was purchased from Scharlau, while Tris (hydroxymethyl) aminomethane was obtained from Research Organics. Sodium dodecyl sulfate (SDS), acrylamide, ammonium peroxodisulfate (APS), glycine, Coomassie Brilliant Blue, bovine serum albumin (BSA), N,N,N′,N′-tetramethylenediamine (TEMED), bromophenol blue, and trypsin were supplied from Merck, while nisin was purchased from Suzhou Hengliang Co. Ltd., Suzhou, China. The 2-mercaptoethanol and HCl were supplied from Riedel-deHaen, Seeize, Germany. Sunset Yellow FCF No. 6 was a gift from coauthor Asad Sayeed (National Foods Ltd., Karachi, Pakistan). Glycerol and takadiastase from Aspergillus oryzae were purchased from Fluka, Steinhein, Denmark. Preparation of Slab Gel The following solutions were first prepared. For solution A, acrylamide (30 g) and bis-acrylamide (0.80 g) were dissolved in 100 mL of double-distilled, deionized water and filtered through Whatman No. 1 filter paper. For solution B, Tris-HCl buffer (1.5M) was prepared by mixing 18.02 g of Tris dissolved in 80 mL of double-distilled, deionized water, and the pH was adjusted to 8.8 by using 1M HCl; the volume was made up to 100 mL. For solution C, Tris (12.114 g) was added to 80 mL of doubledistilled, deionized water; the pH was adjusted to 6.8 with 1M HCl; and the volume was made up to 100 mL with double-distilled, deionized water. For solution D, SDS (10 g) was dissolved in double-distilled, deionized water, and the volume was made up to 100 mL. For solution E, APS (1 g) was dissolved in double-distilled, deionized water, and the volume was made up to 10 mL, while TEMED was used as supplied. The 12.5% slab gel (thickness 0.75 mm) was prepared by using 2.083 mL of solution A, 0.666 mL of solution B, 0.500 mL of solution D, and 0.250 mL of solution E. The mixture was degassed, and 1.66 μL of TEMED was added before use. The gel was left for 15 min to settle. Staking Gel Solutions A, C, D, and E were added in the ratio of 0.416:0.830:0.333:0.166, respectively. The mixture was immediately degassed, and 1.66 μL of TEMED was added before the stacking gel was poured. The wells were prewashed with reservoir buffer after the gel had settled. For the reservoir buffer, Tris (0.9 g), glycine (3.6 g), RESEARCH

An Unusual Twin-His Arrangement in the Pore of Ammonia Channels Is Essential for Substrate Conductance

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.