PMC:103662 / 26911-31269 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/103662","sourcedb":"PMC","sourceid":"103662","source_url":"https://www.ncbi.nlm.nih.gov/pmc/103662","text":"Materials \u0026 methods\n\nProtein expression and detergent extraction\nProteorhodopsin was expressed by E. coli strain UT5600 containing an additional plasmid encoding for the first-reported pR gene (accession #AF279106, obtained from the uncultured proteobacterium EBAC31A08 clone BAC [1]) with an Ara promoter and ampicillin resistance (kindly provided by O. Béjà). Single colonies were selected and grown overnight in LB/amp media (200 ml, 37°C, 300 rpm). This culture was then diluted 10× into several 500-ml cultures. After a further 2 h incubation in the shaker bath, a stock solution of 20% L-arabinose was added, to give a final concentration of 0.2% L-arabinose. This culture was then incubated for 4 h (37°C, 300 rpm). The cells (~20 ml wet volume) were then collected by centrifugation (6000 rpm × 30 min) and washed 3× with 100 mM HEPES, pH 7.1 (buffer A). The cells were then resuspended in buffer A and incubated at 4°C with 50 μg all-trans-retinal (added as a concentrated ethanol solution) for 3 h. The cells were collected by centrifugation (6000 rpm × 30 min), then resuspended in 60 mL buffer A containing 0.3 mg/mL lysozyme, and stirred for 4 h at room temperature. The cells were again collected by centrifugation (6000 rpm × 30 min), then lysed with 20 ml of 20% sodium cholate, pH 7.1 (30 min., 4°C). The cells were centrifuged again (6000 rpm × 30 min), and the supernatants collected. After extracting 3× more with the same cholate solution, the pooled supernatants were diluted 10× with buffer A and centrifuged at 180,000 g for 45 min to collect the membrane pellet. This cholate-washed membrane pellet was then further extracted 3× with 3.0% β-octyl-D-glucoside (OG) in buffer A (30 min, with stirring, 4°C). The pooled supernatants, containing OG-solubilized pR (~15 mg), were then diluted 6× with buffer A.\n\nColumn purification\nThe diluted OG-solubilized membrane extract (10 mg in 300 mL total volume of 0.5% OG) was loaded on a 25 × 1 cm column containing Phenylsepharose™ (6 fast flow high sub; Amersham Pharmacia Biotech). The column was eluted with a 0.5%-2.0% OG gradient in buffer A (300 mL total volume, 0.5 mL flow rate). The pR eluted at an OG concentration of 1.5–2.0%. Fractions having an A280/A546 ratio of 4.0 or lower were pooled (9.5 mg pR recovered in all) and concentrated using Vivaspin™ 20 concentrators having a 5000 MW cutoff (Vivascience, Westford, MA). A portion of the Phenylsepharose™-purified pR (1.5 mg) was diluted to an OG concentration of 0.5% with 0.5 M KCl, 100 mM acetate. It was then loaded on a 10 cm × 1 cm hydroxylapatite (BioGel HTP, BioRad) column and eluted under pressure with a 0–600 mM phosphate gradient (200 mL total volume, flow rate 0.5 ml/min). Fractions with an A280/A546 ratio of 2.5 or lower were pooled and concentrated for subsequent experiments (0.5 mg).\n\nMutagenesis\nMethodology for the site-directed mutagenesis of pR is discussed in detail elsewhere (R. Parthasarathy, T. Caterino, R.A. Krebs, M.S. Braiman, manuscript in preparation). The triple cysteine mutant (pR-TCM) has all three of its native cysteines (Cys-107, Cys-156, and Cys-175) replaced with serines, and was prepared using the same E. coli expression system and purification methods as the wild type.\n\nPolyacrylamide gel electrophoresis\nA 12% discontinuous SDS/polyacrylamide gel was used for molecular weight and purity analysis [17].\n\nFlash photolysis\nTime-resolved UV/vis spectroscopy methods were as described previously [18]. A Phenylsepharose™-purified pR sample was reconstituted into mixed micelles containing 1,2-diheptanoyl-SN-glycero-3-phosphocholine (DHPC), by adding a 1% solution of the short-chain lipid and then removing most of the detergent on a Sephadex G-25 column equilibrated with 1% DHPC in 100 mM NaCl. Proton release and uptake in the aqueous bulk medium were detected from the pR-containing micelles suspended in 1% DHPC, 100 mM NaCl, with 45 μM Cresol Red pH indicator dye. Flash-induced absorbance changes at 580 nm of samples with and without the Cresol Red were subtracted to determine the transient signals due to proton concentration changes. Photoexcitations were performed with 10-ns laser pulses of 3–6 mJ at 500 nm. The time courses in Figs. 3,4 are an average of 40 cycles with the exception of the Cresol Red experiments averaging 100 cycles (Fig. 5, bottom trace) [19-21].","divisions":[{"label":"title","span":{"begin":0,"end":19}},{"label":"sec","span":{"begin":21,"end":1830}},{"label":"title","span":{"begin":21,"end":64}},{"label":"p","span":{"begin":65,"end":1830}},{"label":"sec","span":{"begin":1832,"end":2833}},{"label":"title","span":{"begin":1832,"end":1851}},{"label":"p","span":{"begin":1852,"end":2833}},{"label":"sec","span":{"begin":2835,"end":3247}},{"label":"title","span":{"begin":2835,"end":2846}},{"label":"p","span":{"begin":2847,"end":3247}},{"label":"sec","span":{"begin":3249,"end":3382}},{"label":"title","span":{"begin":3249,"end":3283}},{"label":"p","span":{"begin":3284,"end":3382}},{"label":"title","span":{"begin":3384,"end":3400}}],"tracks":[]}