Chemistry and Physics Faculty Proceedings, Presentations, Speeches, Lectures

Title

Expanding Magnetic Resonance Techniques into Live Whole Cells for Membrane Protein Studies

Event Name/Location

72nd Annual Meeting of the American Scientific Affiliation, Golden, Colorado, July 28-31, 2017

Document Type

Poster

Publication Date

7-2017

Abstract

Membrane proteins may be influenced by environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structure in a native environment are highly desirable. Whole cell DEER studies represent a novel general strategy for precise distance measurements on outer membrane proteins in whole E. coli cells and isolated outer membranes.

In this work, the cobalamin transporter BtuB was overexpressed and spin labelled in whole cells and outer membranes, and interspin distances were measured to a spin labelled cobalamin using pulse EPR. A comparative analysis of the data reveals a similar interspin distance distribution between whole cells, outer membranes and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions for large outer membrane protein complexes in whole cells and native membranes, and provides a method to validate high-resolution structures of membrane proteins in their native environment.

In this study, the concentration of BtuB used corresponds to about 105 copies per cell, which is comparable to the expression level of some endogenous outer membrane proteins. A comparison of data between three different membrane environments indicates that the lipid composition does not significantly influence the loop conformation. These native preparations provide a versatile tool to study membrane proteins and unlike the alternate approaches, does not require the tedious processes of solubilization, purification, and membrane reconstitution of the target protein.

Comments

NIGMS grant #: HM035215; Deutsche Forschungsgemeinschaft grant #: SFB 807

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