Chloroquine (CQ) has been used for decades as the primary chemotherapeutic drug 
for the treatment of malaria. The emergence of drug resistance in Plasmodium 
falciparum has been considered to be because of the excessive use of 
antimalarial drugs worldwide. Moreover, the intense distribution and prevalence 
of chloroquine-resistant strains in endemic regions has aided the incidence of 
more complications to malaria treatment and control. Due to the lack of 
literature that portrays evident molecular mechanisms of drug resistance, it has 
been difficult to understand the drug resistance conferred by Plasmodium 
species. Intensive research on CQ drug resistance has identified the association 
of P. falciparum chloroquine resistance transporter protein (PfCRT), which 
belongs to the drug/metabolite transporter and EamA-like superfamily. 
Additionally, it has shown that K76 T mutation in PfCRT protein has mainly 
attributed to CQ resistance than other mutations. This study deals with the 
development of an in silico model of the PfCRT protein and its interaction with 
the CQ ligand molecule as well as the biochemical and biophysical 
characterization of the transmembrane domain 1 (TMD 1) peptide of the PfCRT 
protein. The physiochemical analysis of the PfCRT protein identified basic 
differences between the wild and mutant forms of the protein, as well as 
identifying the high hydrophobic nature of the mutant-type protein. The tertiary 
structure of the PfCRT protein was predicted and interaction with CQ revealed 
different active pocket binding regions in both the wild and mutant form of 
PfCRT proteins. The CQ2+ molecule interacts with TMD 10 of the wild-type PfCRT 
protein, whereas it interacts with TMD 1 of the mutant-type protein. Studies on 
the TMD 1 peptide revealed the insertion of the peptide in the micelles adopting 
stable alpha-helical structure. Binding studies with the CQ molecule detected 
high binding affinity toward the mutant-type TMD 1 peptide rather than the 
wild-type, thus confirming that the TMD 1 peptide is involved in substrate 
selectivity. Our findings help to characterize the structure of the PfCRT 
protein and the role played by the TMD 1 region in CQ resistance using in silico 
and biochemical approaches. Molecular docking and ligand binding studies confirm 
that TMD 1 is involved in substrate selectivity and aids in CQ efflux, thereby 
contributing to the parasite's CQ drug resistance mechanism.