Chemistry and Physics Faculty Articles

Title

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR

Document Type

Article

Publication Date

2-17-2016

Publication Title

Journal of the American Chemical Society

ISSN

0002-7863

Volume

138

Issue/No.

6

First Page

1844

Last Page

1847

Abstract

An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron–electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. With this approach, structure, function, conformational changes, and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment.

Comments

©2016 American Chemical Society

Additional Comments

Deutsche Forschungsgemeinschaft grant #: SFB 807;; NIGMS grant #: GM035215

DOI

10.1021/jacs.5b13382

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Peer Reviewed

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