Biochemical and biophysical analysis of recombinant Plasmodium falciparum chloroquine resistance transporter (pfcrt)
Paguio, Michelle Fortaleza.
Thesis (Ph.D.)--Georgetown University, 2009.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Malaria caused by the Apicomplexan parasite Plasmodium falciparum is an alarming health problem due to increasing incidence of deaths and growing resistance to antimalarial drugs such as chloroquine. Chloroquine resistance in P. falciparum is primarily determined by PfCRT (P. falciparum chloroquine resistance transporter), a transmembrane protein localized to the digestive vacuolar membrane of the parasite. Bioinformatic studies by Fidock et al. (2000) and Martin & Kirk (2004) suggested that PfCRT belongs to the drug/metabolite transporter superfamily but its mode of action has not yet been fully established. In order to elucidate the role of PfCRT in chloroquine resistance, chloroquine binding and transport were analyzed using membrane preparations from yeast Pichia pastoris expressing PfCRT, as well as proteoliposomes harboring purified protein.; Chloroquine binding was investigated employing crude and purified membranes with either radiolabeled or photoaffinity-labeled chloroquine. Under de-energized conditions, radiolabeling experiments showed PfCRT protein to specifically bind chloroquine with a Kd near 400 nM but did not measurably bind the related drug quinine at physiologically relevant concentrations. Photolabeling studies also demonstrated that chloroquine sensitive and resistant PfCRT isoforms have similar chloroquine binding affinities.; Various investigators have proposed that PfCRT may function either as a channel or an active transporter. Using a novel probe, wherein the fluorescent NBD tag is attached to a terminal chloroquine ethyl group, purified isoforms of PfCRT reconstituted into proteoliposomes, and a novel assay involving extra-proteoliposomal quenching with dithionite, efflux was further analyzed under a variety of conditions. Turnover numbers derived from initial rates of efflux show that the outward movement of CQ via PfCRT is membrane potential-dependent. These data strongly support the hypothesis that PfCRT catalyzes facilitated diffusion of charged CQ.; Two new methods were also developed: a high throughput fluorescence assay for antimalarial drug screening and a convenient scheme for the purification of recombinant PfCRT. The drug assay has contributed to the efficiency and ease of testing numerous compounds while the optimization of purifying recombinant PfCRT has produced a cleaner and more reliable system to investigate its function. These methods significantly aided in understanding the mechanism of chloroquine resistance.
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