Title

G-protein coupled CB2 receptor: Method development for membrane protein NMR

Date of Completion

January 2004

Keywords

Biology, Molecular|Chemistry, Pharmaceutical|Health Sciences, Pharmacy

Degree

Ph.D.

Abstract

The experiment-based three-dimensional structures of G protein coupled receptors (GPCRs) would have greatly aided the understanding of their structure-function relationships, thus facilitating rational ligand design for these biologically and therapeutically important integral membrane proteins. In considering the difficulties to study the GPCR structures by conventional approaches, a “NMR structure from building blocks to 3D assemblies” strategy was proposed and investigated. In order to validate and apply this approach, a comprehensive method development was conducted. ^ The scope of the research work described in this thesis was mainly focused on the method development of GPCR segments expression, purification, and sample preparation. To achieve this objective, cannabinoid receptor subtype CB2 was chosen as a target molecule. Transmembrane helices and multi-helix bundles from CB2 were expressed in E. coli in a high yield, followed by multi-step purification procedures. Expression and purification protocols were developed, including usage of fusion protein over-expression systems, nickel affinity chromatography, chemical or enzymatic cleavage, and reverse phase HPLC. These processes were monitored by electrophoresis, HPLC, mass spectrometry, and circular dichroism spectroscopy. The final products were analyzed and validated by these analytical methods as well. Preliminary NMR studies characterized the prepared CB2 segments, and thus validated the developed methods. ^ With the developed expression and purification schemes, milligrams of uniformly isotope-labeled membrane protein samples were prepared and purified. The samples were sufficient and qualified for membrane protein NMR studies and structure determinations as other continuing projects. The developed methods were standardized, and will be adapted to produce other transmembrane helices and helix bundles of CB2, and those from other GPCRs, for their membrane protein NMR studies and other biophysical studies in the future. ^