​ogic.​ca/​projects/​k2d2/​[34] to evaluate the secondary structure content. Turbidity

Assay Turbidity measurements were taken on a Multiskan Spectrum double-beam spectrophotometer (Thermo Electro Corp.) by using 1 cm matched silica cuvettes at 400 nm. The SUV concentration was 250 μM. The lipid:protein ratio for the turbidity assays was kept at 50:1. Vesicle Internal Content Mixing Small unilamellar vesicles were prepared containing either 5 mM terbium chloride, 50 mM sodium citrate,10 mM Tris/HCl (pH 7.4), or 50 mM sodium dipicolinate (DPA) and 10 mM Tris-HCl (pH 7.4). The vesicles concentration was 100 μM. In both cases, no encapsulated material was removed by gel filtration of the vesicles using Sephadex G-25 (Pharmacia) equilibrated with TH-302 in vitro iso-osmolar 50 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl (pH 7.4). Zero percent and 100% fluorescence (aqueous content mixing) were taken as the intrinsic fluorescence intensity of the Tb/DPA-labeled liposome mixture and the fluorescence obtained after vesicle lysis with 0.2% n-dodecyl maltoside in assay buffer without EDTA as described by Duzgunes et al [35]. Fluorescence measurements were carried out at 25°C using a Molecular

Devices Ilomastat concentration SpectroMAX GeminiEM spectrofluorometer. The extent of vesicles aqueous content mixing was determinated according to the following equation: Where F0 is the value of initial fluorescence of the vesicles, Ft is the value of fluorescence after incubation for t minutes with the protein, and Fmax is the value of fuorescence after addition of 0.2% of n-dodecyl maltoside. Immunoblot analysis Polyclonal anti-YqiC primary antibodies were obtained in mice immunized with purified YqiC. Immobilon-NC Transfer Membranes (Millipore)

containing transferred proteins were blocked in 5% nonfat 17-DMAG (Alvespimycin) HCl milk PBS for 1 h, and incubated with either a 1:200 dilution of polyclonal anti-YqiC or 1:200 anti-MBP mouse polyclonal antibodies. The secondary antibody used was goat anti-mouse IgG (Fc Specific) Peroxidase Conjugate (Sigma) at 1:1000 dilution. Positive signals were detected with Chemiluminiscent ECL Plus Western Blotting Detection System (Amersham Biosciences) on a Storm Image and Detection system (Molecular Dynamics). Cell fractionation Wild-type S. Typhimirium strain was grown in 80 mL LB medium to an OD600 of 1 and harvested by centrifugation at 4000 × g. The pellet was resuspended in 3 ml 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl and mechanically lysed in a FastPrep instrument. Cell debris was removed by centrifugation for 30 min at 8000 × g. Subsequently, membranes were sedimented by ultracentrifugation for 1 h at 100,000 × g (4°C). The pellet was resuspended in a volume equivalent to that of the supernatant. Samples from the supernatant and pellet fraction were analyzed by immunoblotting. Construction of yqiC S. Typhimurium mutant strain Elimination of the yqiC gene was achieved by using Lambda Red-mediated recombination described previously [36].