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Characterization of the Walker A motif of MsbA using site-directed spin labeling electron paramagnetic resonance spectroscopy. Biochemistry 2005 Apr 12;44(14):5503-9

Date

04/06/2005

Pubmed ID

15807544

DOI

10.1021/bi047568v

Scopus ID

2-s2.0-16844381206   41 Citations

Abstract

MsbA is an ABC transporter that transports lipid A across the inner membrane of Gram-negative bacteria such as Escherichia coli. Without functional MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, several functionally important motifs common to ABC transporters are unresolved in the crystal structure. The Walker A domain, one of the ABC transporter consensus motifs that is directly involved in ATP binding, is located within a large unresolved region of the MsbA ATPase domain. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions. MsbA reconstituted into lipid membranes has been evaluated by EPR spectroscopy, and it has been determined that the Walker A domain forms an alpha-helical structure, which is consistent with the structure of this motif observed in other crystallized ABC transporters. In addition, the interaction of the Walker A residues with ATP before, during, and after hydrolysis was followed using SDSL EPR spectroscopy in order to identify the residues directly involved in substrate binding and hydrolysis.

Author List

Buchaklian AH, Klug CS

Author

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

ATP-Binding Cassette Transporters
Bacterial Proteins
Crystallography
Electron Spin Resonance Spectroscopy
Models, Molecular
Mutagenesis, Site-Directed
Spin Labels
jenkins-FCD Prod-400 0f9a74600e4e79798f8fa6f545ea115f3dd948b2