Name: Patrick Seelheim
Diploma / M.Sc degree: Westfälische Wilhelms-Universität Münster, Germany
(September 2009)

PhD Project: Cooperative Effects of the Human ATP-Binding Cassette Transporter ABCC3 in Surfaces-Immobilized Membrane Systems


Homepage: http://www.uni-muenster.de/Chemie.bc/staff/bc-staff.html

Abstract of Research Project

Human ATP-binding cassette (ABC) transporters mediate the active transport of various physiological and xenobiotic substrates across cellular membranes. They are essential for cellular detoxification, maintaining the asymmetry of plasma membranes, the integrity of the blood brain barrier and related to multi-drug resistance of cancer cells. Despite their importance, the actual mechanism of ATP hydrolysis and substrate translocation is still unclear. There is evidence though that the active transporter consists of two subunits that show significant positive cooperativity.

To further elucidate the transport mechanism and the molecular origin of cooperativity, a solid-supported model system for studying ABC transporters is needed. Such a system could be set up by either immobilizing proteoliposomes containing affinity-purified ABC transporters - human ABCC3 in our case - on functionalized surfaces or by direct attachment of the protein to a surface and subsequent assembly of a lipid bilayer by detergent dilution technique. For the first approach ABCC3-containing proteoliposomes doped with a biotinylated anchor lipid can be tethered to streptavidin-functionalized gold surfaces. Alternatively, we try to directly attach tagged subunits of the transporter to structured silicon surfaces bearing adequate functional groups like nickel nitrilo triacetic acid and streptavidin. This would enable us to study the interaction between the protein's subunits. Principal characterization and quality control of the immobilization will be done by quartz crystal microbalance, atomic force microscopy, surface plasmon resonance spectroscopy and ATPase activity assays. The actual transport of known substrates will be monitored by fluorescence based techniques and reveal kinetic parameters including the Hill coefficient as a measure for cooperativity.



Publications

P. Seelheim, H.-J. Galla
Tethered proteoliposomes containing human ABC transporter MRP3: new perspectives for biosensor application based on transmembrane proteins
Biochemical and Biophysical Research Communications 431(3) (2013), 519-523.

P. Seelheim, A. Wüllner, H.-J. Galla
Substrate translocation and stimulated ATP hydrolysis of human ABC transporter MRP3 show positive cooperativity and are half-coupled
Biophysical Chemistry 171 (2013), 31-37.

J. J. Weigand, N. Burford, R. J. Davidson, T. S. Cameron, P. Seelheim
New Synthetic Procedures to Catena-Phosphorus Cations: Preparation and Dissociation of the First cyclo-Phosphino-halophosphonium Salts
J. Am. Chem. Soc. 131 (2009), 17943-17953.


Patrick-Christian Seelheim
eMail: Patrick-Christian Seelheim