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Investigations into the production of integral membrane proteins for solid-state NMR spectroscopy

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dc.contributor.advisor Brown, Leonid Munro, Rachel 2016-01-07T16:12:19Z 2017-01-05T06:00:16Z 2016-01 2016-01-05 2016-01-07
dc.description.abstract 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. en_US
dc.language.iso en en_US
dc.rights Attribution 2.5 Canada *
dc.rights.uri *
dc.subject Research Subject Categories::NATURAL SCIENCES::Chemistry::Molecular biophysics en_US
dc.subject Nuclear Magentic Resonance en_US
dc.subject Integral membrane proteins en_US
dc.subject Isotopic Labeling en_US
dc.subject Adenosine receptor (2A) en_US
dc.subject Anabaena Sensory Rhodopsin en_US
dc.subject Proteorhodopsin en_US
dc.subject Retinal en_US
dc.subject Solid-state NMR en_US
dc.title Investigations into the production of integral membrane proteins for solid-state NMR spectroscopy en_US
dc.type Thesis en_US Biophysics en_US Master of Science en_US Department of Physics en_US
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Attribution 2.5 Canada Except where otherwise noted, this item's license is described as Attribution 2.5 Canada