The class of proteins termed ATP-binding cassette (ABC) transporters is one of the largest found in nature. Their ability, or lack thereof, to move a variety of ligands across a membrane bilayer using energy from ATP is fundamentally important to bacterial physiology and an array of human pathologies. ABC transporters mediate both the import and export of a wide variety of solutes including antibiotics, lipids, chemotherapy agents, sugars, amino acids, salts and metals. MsbA is the ABC transporter for lipid A that is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A, which is an essential component of the outer surface of the cell, within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for structural and functional dynamics studies. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. The goal of this proposal is to elucidate the conformational dynamics that occur in MsbA, a bacterial ABC transporter, upon binding ATP in its nucleotide binding domain and upon recognition and transport of lipid substrates in its helical core, utilizing site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy techniques. In order to address the proposal that the MsbA homodimer undergoes significant conformational rearrangements upon ATP and lipid binding that are essential to its function as a lipid exporter, the following points will be addressed: 1) evaluate the quaternary structure of MsbA reconstituted into lipid membranes; 2) investigate the conformational dynamics of the MsbA dimer upon ATP binding; and 3) investigate the conformational dynamics of the MsbA dimer upon lipid binding. It is anticipated that these studies will produce valuable insights into the local and global structural dynamics of MsbA as it functions in its role as a lipid transporter.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070642-02
Application #
6877100
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
2004-05-01
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
2
Fiscal Year
2005
Total Cost
$225,000
Indirect Cost
Name
Medical College of Wisconsin
Department
Biophysics
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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