"Investigations into the production of integral membrane proteins for solid-state NMR spectroscopy"
This study seeks to probe the challenges associated with heterologous expression and isotopic labeling of microbial rhodopsins and the GPCR adenosine receptor (2A) (A2aR) through biophysical methods including SSNMR, FTIR and Raman spectroscopy. A2aR was transformed into Pichia pastoris, which has previously been shown to be able to cost-effectively produce isotopically-labelled eukaryotic membrane proteins for SSNMR studies; however poor expression resulted in contamination as detected by both FTIR and SSNMR. Next, for Anabaena sensory rhodopsin (ASR) produced in E. coli, we investigated the effect of a full-length construct on both expression and purification. Finally, we showed that the novel biosynthetic production of an isotopically labelled retinal ligand concurrently with its apoprotein proteorhodopsin in E. coli presents a cost effective alternative to de novo synthesis. By using alternately labeled carbon sources (glycerol) we were able to verify the biosynthetic pathway for retinal and assign several new carbon resonances for proteorhodopsin-bound retinal.
Dr. Hermann Eberl, Chair (Department of Mathematics and Statistics)
Dr. Leonid Brown, Advisor (Department of Physics)
Dr. Vladimir Ladizhansky, Advisory Committee (Department of Physics)