PELDOR/DEER Spectroscopy on a Bacterial Membrane Transporter in Native Cellular Environemnts

Abstract

Membrane protein function encompasses local and global motions occurring over a wide range of time scales (from ps to ms), which all could be modulated by the surrounding environment [1, 2]. However, determination of membrane protein structure and dynamics with high resolution in intact cells is yet to be demonstrated. We applied continuous wave and pulsed EPR spectroscopy combined with molecular biology techniques to monitor dynamics and conformation of the outer membrane cobalamin transporter BtuB in intact E. coli cells [3]. BtuB can be overexpressed in E. coli cells up to 105 copies per cell, comparable to the expression level of some endogenous outer membrane proteins. Overexpressed BtuB can be spin labelled and it can bind substrate and undergo conformational changes in live E. coli cells. Further, it is feasible to perform distance measurements using pulsed EPR on BtuB in whole E. coli cells and native outer membranes. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein–protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.1. Mchaourab, H.S., Steed, P.R., Kazmier, K. (2011) Structure 19,1549-1561.2. Freed, D.M., Khan, A.K., Horanyi, P.S., Cafiso D.S. (2011) Biochemistry 18, 8792-803.3. Joseph, B, Sikora, A., Bordignon, E,, Jeschke, G., Cafiso, D.S., Prisner, T.F. (2015) Angew. Chem. Int. Ed. 54, 6196-6199.