“Development of a perdeuteration protocol to study yeast-produced eukaryotic membrane proteins using solid-state NMR spectroscopy”
A number of obstacles exist in the study of eukaryotic membrane protein structure using solid-state NMR. Firstly, although bacterial hosts are often used to produce proteins due to their rapid cell growth, cheap costs, and high expression, there are many difficulties in using bacterial expression systems to produce eukaryotic proteins. Bacteria possess different membrane composition, exhibit improper membrane targeting, and lack the cellular machinery to make native-like post-translational modifications—all of which are important for proper folding of proteins. Another difficulty that arises with studying membrane proteins is their size, the abundance of overlapping peaks, and the broadening of peaks from proton-proton coupling. To address the first roadblock, the yeast system, Pichia pastoris, is commonly used, among others. This system maintains relatively high cell growth rate, high expression and low costs, while offering post-translational modifications and proper folding of eukaryotic membrane proteins. To address the second obstacle, deuteration is used. Deuterons are introduced to replace many of the protons in the sample, reducing the number of overlapping peaks as well as narrowing proton peaks. Compared to the use of deuteration in studying soluble proteins, its use in studying membrane proteins in yeast systems like Pichia pastoris remains relatively unexplored. Using the Pichia pastoris yeast system and the protein Leptosphaeria rhodopsin, this study aims to develop a perdeuteration protocol that addresses the above-mentioned challenges to further advance the use of solid-state NMR in membrane protein structural and dynamics studies.
- Prof. Xiao-Rong Qin, Chair (Department of Physics)
- Prof. Vladimir Ladizhansky, Advisor, (Department of Physics)
- Prof. Leonid Brown, Co-Advisor, (Department of Physics)
- Prof. Steffen Graether, Examination Committee (Dept. of Molecular and Cellular Biology)