The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain–binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding–induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain–binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates

Ninth and Tenth Order Virial Coefficients for Hard Spheres in D Dimensions

We evaluate the virial coefficients B_k for k<=10 for hard spheres in dimensions D=2,...,8. Virial coefficients with k even are found to be negative when D>=5. This provides strong evidence that the leading singularity for the virial series lies away from the positive real axis when D>=5. Further analysis provides evidence that negative virial coefficients will be seen for some k>10 for D=4, and there is a distinct possibility that negative virial coefficients will also eventually occur for D=3.Comment: 33 pages, 12 figure

Antibacterial activity of some selected medicinal plants of Pakistan

<p>Abstract</p> <p>Background</p> <p>Screening of the ethnobotenical plants is a pre-requisite to evaluate their therapeutic potential and it can lead to the isolation of new bioactive compounds.</p> <p>Methods</p> <p>The crude extracts and fractions of six medicinal important plants (<it>Arisaema flavum</it>, <it>Debregeasia salicifolia</it>, <it>Carissa opaca</it>, <it>Pistacia integerrima</it>, <it>Aesculus indica</it>, and <it>Toona ciliata</it>) were tested against three Gram positive and two Gram negative ATCC bacterial species using the agar well diffusion method.</p> <p>Results</p> <p>The crude extract of <it>P. integerrima </it>and <it>A. indica </it>were active against all tested bacterial strains (12-23 mm zone of inhibition). Other four plant's crude extracts (<it>Arisaema flavum</it>, <it>Debregeasia salicifolia</it>, <it>Carissa opaca</it>, and <it>Toona ciliata</it>) were active against different bacterial strains. The crude extracts showed varying level of bactericidal activity. The aqueous fractions of <it>A. indica </it>and <it>P. integerrima </it>crude extract showed maximum activity (19.66 and 16 mm, respectively) against <it>B. subtilis</it>, while the chloroform fractions of <it>T. ciliata </it>and <it>D. salicifolia </it>presented good antibacterial activities (13-17 mm zone of inhibition) against all the bacterial cultures tested.</p> <p>Conclusion</p> <p>The methanol fraction of <it>Pistacia integerrima</it>, chloroform fractions of <it>Debregeasia salicifolia </it>&<it>Toona ciliata </it>and aqueous fraction of <it>Aesculus indica </it>are suitable candidates for the development of novel antibacterial compounds.</p

Buckling Under Pressure: Curvature-Based Lipid Segregation and Stability Modulation in Cardiolipin-Containing Bilayers

Mitochondrial metabolic function is affected by the morphology and protein organization of the mitochondrial inner membrane. Cardiolipin (CL) is a unique tetra-acyl lipid that is involved in the maintenance of the highly curved shape of the mitochondrial inner membrane as well as spatial organization of the proteins necessary for respiration and oxidative phosphorylation. Cardiolipin has been suggested to self-organize into lipid domains due to its inverted conical molecular geometry, though the driving forces for this organization are not fully understood. In this work, we use coarse-grained molecular dynamics simulations to study the mechanical properties and lipid dynamics in heterogeneous bilayers both with and without CL, as a function of membrane curvature. We find that incorporation of CL increases bilayer deformability and that CL becomes highly enriched in regions of high negative curvature. We further show that another mitochondrial inverted conical lipid, phosphatidylethanolamine (PE), does not partition or increase the deformability of the membrane in a significant manner. Therefore, CL appears to possess some unique characteristics that cannot be inferred simply from molecular geometry considerations

Assays for AAC integration into liposomes.

<p>A) Proteoliposomes containing the indicated radiolabeled proteins were incubated in the presence and absence of 100 mM Na₂CO₃ (pH 11.5). Following carbonate extraction, pellets and TCA-precipitated supernatants were resolved by SDS-PAGE. The percentage of protein per sample has been indicated. B) Site-specific labeling of AAC monocysteine variants with thiol-reactive NBD. AAC constructs with single cysteine residues on either the second cytosolic loop (AAC L219C, top panel) or transmembrane segment four (AAC L194C, bottom panel) were translated in the presence or absence of liposomes as indicated and subjected to a labeling reaction time course with IANBD. After quenching the reaction, samples were resolved by SDS-PAGE and the relative extent of labeling was quantified by in-gel fluorometry. The mean labeling efficiencies of four independent measurements is shown with standard deviations. Where indicated, the extent of labeling between samples with and without liposomes was significantly different (p<0.01 [**] and p<0.001 [***]) based on two-tailed t-tests.</p

<i>In vitro</i> translated AAC in proteoliposomes is transport competent.

<p>Liposomes (A) or AAC-containing proteoliposomes (B, C) pre-loaded with ATP were incubated in the translation reaction to allow transport to occur. Vesicles were then purified by size-exclusion chromatography to remove unencapsulated nucleotide and assayed for total ATP content by luminescence immediately after disruption with detergent (0.2% triton X-100). A) Luminescence measurements of ATP-loaded liposomes pre-incubated with buffer only (white bar) or with detergent (hatched bar) registered a higher luminescence signal (p<0.0001 [****], two-tailed t-test) following disruption of the vesicles to release ATP. B) AAC-proteoliposomes with encapsulated ATP were prepared in the presence or absence of CAT as indicated and subjected to pretreatment with buffer only (white bars) or detergent (hatched bars) prior to luminescence measurements. The internal ATP content of proteoliposomes without CAT was significantly lower than samples prepared with CAT (p<0.01 [**], two-tailed t-test). The presence of CAT itself had no effect on the luminescence readings. C) AAC-proteoliposomes were prepared as in panel B, then incubated with buffer only (white bar) or 0.2 mM ADP (hatched bar), purified by a second round of gel filtration, and assayed for total luminal ATP content after detergent disruption. The internal ATP content of proteoliposomes subjected to ADP incubation was significantly lower than samples incubated with buffer only (p<0.01 [**], two-tailed t-test). All relative luminescence intensity measurements are means from a minimum of three independent experiments with standard deviations.</p

Cell-free translation and purification of AAC-containing proteoliposomes.

<p>A) Schematic illustration of the experimental process. [³⁵S]AAC is translated in a wheat germ-based system in the presence of SUVs and the sample is subjected to SGU. Buoyant proteoliposomes remain at the top of the gradient, whereas aggregated protein and ribosomes from the translation reaction pellet. B) The extent of AAC integration depends on liposome concentration. [³⁵S]AAC was translated in the presence of variable SUV concentrations as indicated and samples from each fraction were resolved by SDS-PAGE. Fractions 1–4 indicate fractions collected from top to bottom of the gradient. Normalized band intensities are shown below each lane of the gel.</p