Defining Topologically Oriented Transmembrane Helix Contacts in Integral Membrane Protein EmrE by a Modified Two-Hybrid Approach
Date
2014-04-01
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Abstract
Study of integral membrane proteins is comparatively difficult in comparison to soluble proteins, requiring more specialized techniques. Here is presented an application of the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) method to study oligomerization in membrane proteins applied to model integral membrane multidrug transporter EmrE. The system was unable to observe dimerization between full length monomers of EmrE, likely due to the preferential antiparallel nature of EmrE dimers. Further studies required large numbers of samples, so the Miller assay was adapted to work in a small volume microtitre plate format. Using this new higher throughput assay, constructs of individual EmrE transmembrane helices (TMH) were utilized to perform contact mapping of EmrE dimer structure. The results were compiled to construct a transmembrane helix interaction structure. This structure obtained is novel and identifies inconsistencies from previous studies.
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Biochemistry
Citation
Burt, J. (2014). Defining Topologically Oriented Transmembrane Helix Contacts in Integral Membrane Protein EmrE by a Modified Two-Hybrid Approach (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/25539