AbstractAdenosine triphosphate binding cassette (ABC) transporters form a ubiquitous superfamily of integral membrane proteins involved in the translocation of substrates across membranes. Substrate translocation is powered by ATP. In this study, four medically important classes of ABC transporters were chosen for structural studies: namely, subfamily B, member 5 (ABCB5), subfamily B, member 6 (ABCB6), subfamily G, member 1 (ABCG1), subfamily G, member 4 (ABCG4). A bioinformatics approach was used for ortholog selection of representative ABC transporters to improve the process of structure determination. Selected orthologs were expressed in Saccharomyces cerevisae with polyhistidine affinity and Green Fluorescent protein identification tags and purified in the presence of the detergent, n-Dodecyl-ÃÂ²-D-Maltoside. A battery of tests was applied to explore the quality of purified proteins. An assay using a thiol-specific dye, and intrinsic protein fluorescence was used to assess the thermal stability of proteins. ATPase activity, measured in a detergent environment, showed that all of the proteins hydrolysed ATP. A membrane thermal shift experiment demonstrated that mouse ABCG4 was stabilized by tyrosine kinase inhibitors. Mouse ABCG4 was also the subject of cryoEM imaging. Taken together, this project throws light on the use of computational tools for target selection before conducting structural studies. All target proteins were successfully expressed in yeast. Protein purification showed mixed results; however, all proteins could be enriched. All purified protein targets were functionally active. The assays could form a platform for screening compounds that stabilise the proteins. Stabilized proteins could subsequently go into crystal trials.
|Date of Award||1 Aug 2023|
|Supervisor||Richard Collins (Supervisor) & Stephen Prince (Supervisor)|
- Saccharomyces cerevisae
- cryo-EM etc.
- ABC transporters