Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine.

Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine. Research Abstract Details 

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Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine. Abstract Text:

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.

Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine. Publishing Authors By Initials

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Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Biochemistry

VOLUME: 47

Page Numbers: 10394-406

Journal Abbreviation: Biochemistry

ISSN: 1520-4995

DAY: 4

MONTH: 09

YEAR: 2008

Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine. Information

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LANGUAGE: eng

NlmUniqueID: 370623

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Grant and Affiliation Information for Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine.

AFFILIATION: Program in Tumor Biology, Georgetown University, Washington, DC 20057, USA.

Country: United States

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MEDLINETA: Biochemistry

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