Directionally Drilled Raw Water Intakes, Grand Forks, North Dakota

The City of Grand Forks, North Dakota obtains drinking water from both the Red River and Red Lake River through a system of raw water intakes, shallow pipelines and pump stations. During flood events, the City often loses access to the system. In addition, the banks of the rivers are subject to land sliding, which can easily damage the shallow intakes. This proved particularly true during the record flood event in 1997, and resulted in the design of a new setback levee system by the U.S. Army Corps of Engineers. As a result, the City decided to construct a new gravity raw water intake system inland of the future levees. The design had to address the installation of pipe through soft and weak clay in a known landslide area to depths of up to 80 feet. Horizontal directional drilling (HDD) was chosen as the means of construction. Design issues associated with HDD included the potential for squeezing ground at the deepest sections of the alignment, the potential for hydraulic fracturing beneath the river bottom and at the exit points, river taps, penetrations though a large-diameter caisson pump station. Additional construction issues included bore accuracy and grade to handle design curves, control of squeezing ground at the caisson penetrations, and control of the bore annulus as a potential flow path for river water during construction

S. aureus MscL Is a Pentamer In Vivo but of Variable Stoichiometries In Vitro: Implications for Detergent-Solubilized Membrane Proteins

Detergent-induced rearrangements of membrane-protein subunits explain why two MscL channel stoichiometries have been resolved by X-ray crystallography - but S. aureus MscL is truly a pentamer in vivo

The Crystal Structure of Zn(II)-Free Treponema pallidum TroA, a Periplasmic Metal-Binding Protein, Reveals a Closed Conformation

We previously demonstrated that Treponema pallidum TroA is a periplasmic metal-binding protein (MBP) with a distinctive alpha-helical backbone. To better understand the mechanisms of metal binding and release by TroA, we determined the crystal structure of the apoprotein at a resolution of 2.5 Å and compared it to that of the Zn(II)-bound form (Protein Data Bank accession code 1toa). apo-TroA shows a conformation even more closed than that of its Zn(II)-bound counterpart due to a 4° tilt of the C-terminal domain (residues 190 through 308) about an axis parallel to the poorly flexible backbone helix. This domain tilting pushes two loops (residues 248 through 253 and 277 through 286) towards the metal-binding site by more than 1 Å, resulting in an unfavorable interaction of I251 with D66. To avoid this contact, D66 shifts towards H68, one of the four Zn(II)-coordinating residues. The approach of this negative charge coincides with the flipping of the imidazole side chain of H68, resulting in the formation of a new hydrogen bond. The conformational change of H68, along with a slight rearrangement of D279, a C-terminal domain Zn(II)-coordinating residue, distorts the metal-binding site geometry, presumably causing the release of the bound metal ion. Ligand binding and release by TroA, and presumably by other members of the MBP cluster, differs from the “Venus flytrap” mechanism utilized by bacterial nonmetal solute-binding receptors

The <i>S. aureus</i> MscL stoichiometry change is reversible.

<p>(A) The oligomeric state of SaMscL in 4 mM LDAO was determined using SEC-MALS, which measured a protein mass of 61.3 kDa, consistent with a tetrameric channel (top chromatogram). (B) The SaMscL-LDAO sample was exchanged into C₈E₅, and the following day the oligomeric state of SaMscL-C₈E₅ sample was measured by SEC-MALS. Analysis of the SEC-MALS results for the SaMscL-C₈E₅ sample showed a protein mass of 74.4 kDa, consistent with the channel in a pentameric state.</p

LDAO stabilizes a MscL tetrameric oligomeric state as measured by crosslinking, SEC-MALS, and Sedimentation Velocity Centrifugation.

<p>(A) Western blot analysis of DSS-treated SaMscL solubilized with either Triton-X100 or LDAO yield different quantities of tetramers and pentamers. (B) A representative SEC-MALS experiment of SaMscL in LDAO is shown with the UV chromatogram colored blue and the 90° light scattering chromatogram colored red. The total, SaMscL, and LDAO masses are shown as black, green, and orange lines across the elution peak, respectively. The mass of the SaMscL monomer is 14.4 kDa and the tetramer mass is 57.8 kDa. (C) SaMscL in 4 mM LDAO was subjected to sedimentation velocity centrifugation at 50,000 rpm, and the data were analyzed using the noninteracting “species model” of SEDPHAT. Four species, including the detergent micelles, were analyzed. The molar mass of the dominant sedimenting species was 62.5 kDa. In the <i>upper part</i>, the individual data points are depicted as circles, and the best-fit model to those data is shown as lines. The data and fit lines are color coded by color: Violet for the earliest scans, then progressing through indigo, blue, green, yellow, orange, and red as the scans go further forward in time. For clarity only every other scan used in the data analysis is shown. In the <i>lower part</i>, the residuals between the data points and the fitted line are shown and color coded as above.</p

Purified <i>S. aureus</i> MscL is pentameric by equilibrium sedimentation centrifugation and SEC-MALS.

<p>Sedimentation equilibrium centrifugation was performed on 40 µM SaMscL in the neutrally buoyant detergent C₈E₅. The upper two panels in (A) and (B) show scans from three different rotor speeds monitored at either 250 nm (A) or 280 nm (B). The lines represent fits to the data of a single species model that yield a calculated protein mass of 71.2 kDa with a local rmsd of 0.005799 (A) and 0.005059 (B) (SaMscL monomer mass is 14.4 kDa; pentamer mass is 72.2 kDa). The lower two panels in (A) and (B) show the residuals from the data fitting. A representative SEC-MALS experiment of SaMscL in C₈E₅ is shown in (C) with the UV chromatogram colored blue and the 90° light scattering chromatogram colored red. The total computed mass is shown as the black line across the elution peak, with the SaMscL and C₈E₅ mass shown in green and orange, respectively.</p

<i>E. coli</i> and <i>S. aureus</i> MscL are pentameric multimers by crosslinking.

<p>Western blot analysis of DSS-treated <i>E. coli</i>-MscL (Eco) and <i>S. aureus</i>-MscL (Sa) expressed with either the pET21a (pET) or pB10 vector show five distinct bands at both expression levels. Molecular weight markers are shown to the left of the blot and the approximate location of monomers (1×) to pentamers (5×) is shown between the blots. The right-hand blot shows a separate experiment where the majority of the EcoMscL and the SaMscL protein are in the pentameric and monomeric forms.</p

Primers on Molecular Pathways: Bicarbonate Transport by the Pancreas

The pancreas has both endocrine and exocrine functions. As an endocrine organ, stimulation of the pancreatic β-cells results in insulin secretion to control systemic glucose levels. The exocrine function of the pancreas and the need for alkaline pancreatic secretion (pH 8.0–8.5) have been appreciated for more than 40 years. Yet, our knowledge of the cellular mechanisms (signaling, transporters and channels) which accomplish these critical functions has evolved greatly. In the mid-1990s, basolateral Na-bicarbonate (HCO3−) uptake by NBCe1 (Slc4a4) was shown to be critical for the generation of approximately 75% of stimulated HCO3− secretion. In the last 10 years, several new HCO3− transporters in the Slc26 family and their interaction with the cystic fibrosis transmembrane conductance regulator-chloride channel have elucidated the HCO3− exit step at the ductal lumen. Most recently, both IRBIT (inositol 1,4,5-trisphosphate receptor-binding protein) and WNK [with no lysine (K)] kinase have been implicated as additional HCO3− secretory controllers