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Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles
Published on: December 31, 2019
Molecular basis for substrate-dependent transmembrane signaling in an outer-membrane transporter
Stephen M Lukasik1, K W David Ho, David S Cafiso
1Chemistry and Biophysics Programs, Department of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA.
Journal of Molecular Biology
|June 9, 2007
Summary
Substrate binding to the Escherichia coli vitamin B12 transporter (BtuB) triggers a conformational change. This study reveals how specific residue interactions mediate this transmembrane signaling event.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Transmembrane signaling is crucial for cellular processes.
- The Escherichia coli vitamin B12 transporter (BtuB) exemplifies substrate-dependent signaling.
- Understanding these mechanisms is key to cellular regulation.
Purpose of the Study:
- To investigate the molecular interactions mediating substrate-dependent transmembrane signaling in the BtuB transporter.
- To elucidate the role of specific charged residues in BtuB's conformational changes.
Main Methods:
- Utilized site-directed spin labeling (SDSL) to monitor protein conformational dynamics.
- Employed a two-mutant cycle analysis to quantify inter-residue interaction free energies.
- Combined SDSL with thermodynamic analysis for detailed mechanistic insights.
Main Results:
- Identified critical roles for charged residues D316 and R14 in the BtuB structural transition.
- Demonstrated that substrate binding reduces the interaction free energy between D316 and R14.
- Showed this reduction triggers the unfolding of the BtuB energy coupling motif.
Conclusions:
- Substrate binding initiates a cascade leading to energy coupling motif unfolding via specific residue interactions.
- The combined SDSL and mutant cycle analysis is a powerful approach for studying membrane protein signaling.
- This work provides a molecular basis for transmembrane signaling in BtuB.
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