Isotopic labelling strategies for the production of membrane proteins for solid-state NMR

Speaker

Rachel Munro

Abstract

Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging biophysical technique which has been useful in studying the structure of integral membrane proteins such as microbial rhodopsins. This technique requires the incorporation of isotopically-labelled atoms into the protein which usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all carbon and nitrogen sources are isotopically labeled. This process requires optimization as to obtain high yields of homogenously structured protein in order to achieve adequate signal-to-noise for in-depth analysis.

Microbial rhodopsins such as proteorhodopsin and Anabaena sensory rhodopsin have been extensively studied using ssNMR. The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, the retinal binding pocket is of significant interest for ssNMR assignments. Unfortunately, the de novo organic synthesis of an isotopically-labelled retinal is cost-prohibitive in large scale expression. Previously, the biosynthesis of a retinal precursor, β-carotene, has been introduced into many different organisms. This system has been extended to the E. coli expression strain BL21. We have shown 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 organic synthesis. By using fully 13C-labelled glucose as the sole carbon source, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal.

In continuation of the success seen in microbial rhodopsins, we have developed an optimized growth protocol for eukaryotic membrane proteins in the yeast strain Pichia pastoris. Previous work at the university of Guelph has shown this host to be capable of producing membrane proteins with excellent resolution when analysed with ssNMR. However, some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression even in their native host. This can make producing a sample for ssNMR in an economic fashion extremely difficult as growth in minimal media adds additional strain on expression hosts. Our new growth protocol has been shown to increase the yield of full-length, isotopically-labeled hAQP2 ten-fold relative to the previously used protocol resulting in excellent resolution when analysed by two-dimensional ssNMR spectroscopy.