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Assay development for studies of G protein-coupled receptors at the single-molecule level

Olov Wahlsten (Institutionen för teknisk fysik)
Göteborg : Chalmers University of Technology, 2014. - 40 s.
[Licentiatavhandling]

G protein-coupled receptors (GPCRs), also known as seven transmembrane (7TM) receptors, is the largest family of cell surface receptors. These receptors play a key role in transmitting a wide variety of signals across the cell membrane and are involved in physiological processes such as sensory transduction, cell-cell communication, neuronal transmission, and hormonal signaling. For this reason the GPCRs are considered as one of the most pertinent targets for design and development of novel therapeutic compounds, a statement further accentuated by the fact that more than half of the therapeutic agents currently on the market target GPCRs. In this thesis two methods with single molecule sensitivity have been developed for characterization of biomolecular interactions with transmembrane proteins. Single GPCR sensitivity was enabled using total internal reflection fluorescence (TIRF) microscopy, without labeling the GPCR, but instead the lipid membrane of the vesicles containing the GPCR. In the first project the interaction between the GPCR CXCR3 and two of its natural chemokine ligands (CXCL10 and CXCL11) was investigated with the intention to make the developed assay a complementary tool in drug candidate screening. The chemokine ligand (CXCL10) was immobilized on a supported lipid bilayer with polyethylene glycol (PEG) chains to minimize unspecific binding. Vesicles derived from membranes of cells over-expressing CXCR3 were fluorescently labeled by sonicating them together with synthetic fluorescently labeled vesicles. Addition of these labeled native membrane vesicles to the functionalized surface enabled characterization of the ligand-receptor interaction via TIRF mode imaging. Additionally, preliminary results suggest that the method does not require over-expression of the GPCR, which is a major advantage for studying this class of sensitive and low-abundant type of membrane proteins. In the second project a colocalization assay was developed to confirm a specific interaction between a virus particle and a transmembrane protein. The assay is thought to be advantageous due to the facile elimination of unspecific interactions and the possibility to control the number of binding sites on the surface. These advantages in the context of developing robust, cheap and generic assays for future drug discovery are further analyzed and discussed.

Nyckelord: GPCR, TIRF microscopy, QCM-D, pharmacodynamics, drug discovery, equilibrium fluctuation analysis, kinetics, colocalization



Denna post skapades 2014-11-10. Senast ändrad 2014-11-10.
CPL Pubid: 205510

 

Institutioner (Chalmers)

Institutionen för teknisk fysik (1900-2015)

Ämnesområden

Biofysik

Chalmers infrastruktur

Examination

Datum: 2014-12-04
Tid: 10:00
Lokal: PJ-salen, Origo norra, plan 4