Defining Topologically Oriented Transmembrane Helix Contacts in Integral Membrane Protein EmrE by a Modified Two-Hybrid Approach

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.

Name thesis:degree_name

Master of Science (MSc)

Discipline thesis:degree_discipline

Biological Sciences

Grantor dc:publisher.institution

Graduate Studies

Year dc:date.issued

2014

Author dc:creator

• Burt, Jason, Campbell

Advisor dc:contributor.advisor

• Turner, Raymond Joseph

• Biochemistry

Statement dc:rights

• University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.

Language dc:language.iso

eng

DOI

http://dx.doi.org/10.11575/PRISM/25539

OAI identifier oai:identifier

oai:ucalgary.scholaris.ca:11023/1397