No abstract

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of ϳ10 mg/liter bacterial culture. Glutaraldehyde crosslinking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K m ϳ80 M for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

Apolipoprotein A-I (apoA-I, 243 amino acids) is the major protein of high-density lipoproteins (HDL) that plays an important structural and functional role in lipid transport and metabolism. The central region of apoA-I (residues 60−183) is predicted to contain exclusively amphipathic α-helices formed from tandem 22-mer sequence repeats. To analyze the lipid-binding properties of this core domain, four terminally truncated mutants of apoA-I, Δ(1−41), Δ(1−59), Δ(1−41,185−243), and Δ(1−59,185−243), were expressed in baculovirus infected Sf-9 cells. The effects of mutations on the ability of apoA-I to form bilayer disk complexes with dimyristoyl phosphatidylcholine (DMPC) that resemble nascent HDL were analyzed by density gradient ultracentrifugation and electron microscopy (EM). The N-terminal deletion mutants, Δ(1−41) and Δ(1−59), showed altered lipid-binding ability as compared to plasma and wild-type apoA-I, and in the double deletion mutants, Δ(1−41, 185−243) and Δ(1−59, 185−243), the lipid binding was abolished. Thermal unfolding of variant apoA-I/DMPC complexes monitored by circular dichroism (CD) showed hysteresis and a shift in the melting curves by about −12 °C upon reduction in the heating rate from 1.0 to 0.067 K/min. This indicates an irreversible kinetically controlled transition with a high activation energy E a = 60 ± 5 kcal/mol. CD and EM studies of the apoA-I/DMPC complexes at different pH demonstrated that changes in the net charge or in the charge distribution on the apoA-I molecule have critical effects on the conformation and lipid-binding ability of the protein.