Abstract
DnaA protein, the initiator protein of E. coli chromosomal replication, can be rejuvenated from an inactive ADP form to active ATP-DnaA protein by acidic phospholipids in a fluid bilayer. Cross-linking studies with the photoactivable phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H- diazirin -3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine reveal insertion of DnaA protein into the hydrophobic region of the bilayer; this insertion is accompanied by membrane-mediated dissociation of the tightly bound allosteric nucleotides ADP and ATP. Photolabeling of DnaA protein occurred with membrane properties that resembled those needed for reactivation of ADP-DnaA protein; efficient labeling of DnaA protein was observed only when the lipid analog was incorporated into anionic vesicles and the temperature during treatment was above the gel to liquid crystalline phase transition. Predominant hydrophobic photolabeling was localized within a single region of DnaA protein, a region that contains putative amphipathic helices and has been shown to contain information essential for functional interaction with membranes.
Highlights
Regulation of DnaA protein activity is thought to play a vital role in controlling initiation of chromosomal replication in Escherichia coli [1, 2]
Nucleotide-bound DnaA protein was incubated with the vesicles and exposed to light to cross-link the lipid analog with regions of the protein that had inserted into the hydrophobic portion of the membrane bilayer
Peptides that did not contain radiolabel were sought, such that the complete sequence of DnaA protein was represented. These studies, which employed a lipophilic photoreagent, indicate that an essential step in the membrane activation of DnaA protein is the insertion of a distinct domain of the protein into the hydrophobic region of the lipid bilayer
Summary
Materials—Sources were as follows: HEPES, Tricine, CAPS, ATP, and CNBr (Sigma); [␣-32P]ATP (3000 Ci/mmol), NEN Life Science Products; Coomassie Brilliant Blue R-250 and G-250, Bio-Rad; transparanaric acid, Molecular Probes; 2-(2-nitrophenylsulfenyl)-3-methyl3Ј-bromoindolenine (BNPS-skatole), Pierce; 1-stearoyl-2-oleoyl-snglycero-3-[phospho-rac-[1-choline]] and 1-stearoyl-2-oleoyl-sn-glycero3-[phospho-rac-[1-glycerol]], Avanti Polar Lipids, Inc.; trypsin, chymotrypsin, subtilisin, endoproteinase GluC, endoproteinase LysC, and endoproteinase ArgC, Boehringer Mannheim; type HA nitrocellulose filters, Millipore Corp.; Problott PVDF membrane, Applied Biosystems. For phospholipid-mediated release, ATP-DnaA protein was incubated (10 min, temperature as indicated) with small unilamellar vesicles prior to the filtration through nitrocellulose. Photolysis—For [125I]TID-PC/16 photoactivation, reaction mixtures in Eppendorf tubes were irradiated for 30 s at room temperature (unless otherwise indicated) in a Pyrex vessel mounted approximately 10 cm from a Suss LH 1000 lamphouse (Karl Suss, Waterbury Center, VT) equipped with an Osram HBO 350-watt short arc high pressure mercury lamp (30 milliwatts1⁄7cmϪ2). Under these conditions, greater than 90% of the reagent was photolyzed.
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