{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T04:21:53Z","timestamp":1747196513613,"version":"3.40.5"},"reference-count":56,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2024,8,15]],"date-time":"2024-08-15T00:00:00Z","timestamp":1723680000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Health Malaysia Research Grant","award":["(NMRR-19-466-47199 and NMRR-21-1175-60416)"],"award-info":[{"award-number":["(NMRR-19-466-47199 and NMRR-21-1175-60416)"]}]},{"name":"Universiti Teknologi MARA Geran Penyelidikan MyRA","award":["(600-RMC\/GPM LPHD 5\/3 (115\/2021))"],"award-info":[{"award-number":["(600-RMC\/GPM LPHD 5\/3 (115\/2021))"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Malaria, an ancient mosquito-borne illness caused by <jats:italic>Plasmodium<\/jats:italic> parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the <jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic> Kelch 13 (<jats:italic>PfK13<\/jats:italic>) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate <jats:italic>PfK13<\/jats:italic> recombinant proteins incorporating of two specific SNPs mutations, <jats:italic>PfK13<\/jats:italic>-V494I and <jats:italic>PfK13<\/jats:italic>-N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of <jats:italic>PfK13<\/jats:italic> mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between <jats:italic>PfK13<\/jats:italic>-V494I and <jats:italic>PfK13<\/jats:italic>-N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg\/ml for <jats:italic>PfK13<\/jats:italic>-WT, 0.18 mg\/ml for <jats:italic>PfK13<\/jats:italic>-V494I, and 0.28 mg\/ml for <jats:italic>PfK13<\/jats:italic>-N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants <jats:italic>PfK13<\/jats:italic>-N537I (83 a.u.) and <jats:italic>PfK13<\/jats:italic>-V494I (143 a.u.) compared to <jats:italic>PfK13<\/jats:italic>-WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between <jats:italic>PfK13<\/jats:italic> protein mutants with ART. This shows that the <jats:italic>PfK13<\/jats:italic> mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R.<\/jats:p>","DOI":"10.1371\/journal.pone.0306975","type":"journal-article","created":{"date-parts":[[2024,8,15]],"date-time":"2024-08-15T17:27:11Z","timestamp":1723742831000},"page":"e0306975","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of the binding interactions between Plasmodium falciparum Kelch-13 mutant recombinant proteins with artemisinin"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2638-3789","authenticated-orcid":true,"given":"Noorazian","family":"Md. 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