|Title:||To elucidate the structure and function of P. falciparum chloroquine resistance transporter (PfCRT) using the substituted-cysteine accessibility method (SCAM)|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xxi, 189 p. : ill. ; 30 cm.|
|Abstract:||Malaria is a major public health problem in the world. Using multiple site-directed mutagenesis, cysteine-less pfcrt on Dd2 genetic background and thirty-one single cysteine substituted pfcrt within TMD1 and TMD4 were constructed. Twenty one substituted PfCRT within TMD1 and seven in TMD4 were expressed successfully in Pichia pastoris. The microsomes isolated from Pichia pastoris were used to determine the transport activities of PfCRT. Cysteine substitution of obligate amino acids of PfCRT affects the function of PfCRT. Modified by MTSES, Dd2-Cys-less-T76C PfCRT microsomes can accumulate more CQ when the concentration of MTSES increases. Meanwhile, MTSET and MTSEA lead to a decrease in specific CQ accumulation as the concentration of these two MTS reagents increase. This suggests that the charged 76th amino acid affects the CQ accumulation. Substrate protection of MTS reagents modification was carried out in the presence of non-radiolabelled CQ. In these assays, only the 76th showed protection effects. This result strongly suggests that the 76th is a binding site of CQ in PfCRT. Reconstituted proteoliposomes were used to study the PfCRT. Dd2-Cys-less reconstituted proteoliposomes have significant specific CQ accumulation activities. Modification of Dd2-Cys-less-T76C by MTSES demonstrated a higher relative activity and showed that the 76th, 73rd and 67th are accessible to MTS. In the CQ protection assay, CQ impedes the 76th from modification by MTSES and the 76th amino acid of PfCRT may be involved in the position where MTSET modification takes place. In the modification of TMD4 by MTSET, only S163C displays a lower relative activity. This suggests that inducing a positive charge at the 163rd may decrease CQ accumulation. Our results showed that the CQ accumulation ability of the 163rd was not affected by CQ protection. This suggests that the 163rd is different from the 76th, which may be a CQ binding site within PfCRT.|
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