Biosynthetic isotopic labelling strategies for the production of membrane proteins for solid-state Nuclear Magnetic Resonance spectroscopy
Solid-state Nuclear Magnetic Resonance (SSNMR) is an emerging biophysical technique which has been useful in studying the structure and dynamics of integral membrane proteins. This technique requires the incorporation of isotopically-labelled atoms into the protein. This thesis presents strategies for the expression of two classes of integral membrane proteins for SSNMR: microbial rhodopsins and human aquaporins. Firstly, a novel protocol for biosynthetic production of an isotopically labeled retinal ligand concurrently with an apoprotein in E. coli as a cost-effective alternative to the de novo organic synthesis. Detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments. To demonstrate the applicability of this system, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal by using fully 13C-labeled glucose as the sole carbon source. Furthermore, we demonstrated that this biosynthetically produced retinal can be extracted from E. coli cells by applying a hydrophobic solvent layer to the growth medium and reconstituted into an externally produced opsin of any desired labeling pattern. Proteins with lower yields when produced in recombinant organisms can be difficult to express in an economically feasible fashion for SSNMR. We have developed an optimized growth protocol in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy. S256D and S256A hAQP2 mutants, targeting key phosphorylation site, were produced and analysed. The phospho-mimic, S256D-hAQP2, showed an increased yield and more persistent oligomerization as compared to S256A. FTIR, functional assays, and SSNMR indicated that phosphorylation of Ser256 does not affect the transmembrane domain of hAQP2. Furthermore, S256D-hAQP2 showed increased spectral coverage as compared to WT-hAQP2, which makes it an excellent target for more in-depth analysis by SSNMR.