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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling
Published on: May 29, 2011
Conformational mobility of immobilized proteins
Ruin Moaddel1, Irving W Wainer
1Gerontology Research Center, National Institutes in Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA. moaddelru@grc.nia.nih.gov
Journal of Pharmaceutical and Biomedical Analysis
|November 11, 2006
Summary
Immobilized cellular receptors and transporters on chromatography columns retain their ability to change shape in response to drugs or co-factors. This allows for a comprehensive characterization of drug interactions and protein conformations.
Area of Science:
- Biochemistry
- Chromatography
- Pharmacology
Background:
- Cellular receptors and transporters are crucial drug targets.
- Understanding their conformational changes is vital for drug discovery.
- Immobilizing these proteins for study presents unique challenges.
Purpose of the Study:
- To create and validate immobilized receptor and drug transporter columns.
- To investigate ligand- and co-factor-induced conformational changes in immobilized proteins.
- To assess the utility of these columns for drug discovery and characterization.
Main Methods:
- Immobilization of cellular membrane fragments onto silica and glass capillary supports.
- Development of immobilized nicotinic receptor and P-glycoprotein transporter columns.
- Application of frontal affinity and non-linear chromatography techniques.
Main Results:
- Immobilized nicotinic receptors demonstrated agonist-induced conformational shifts (resting to desensitized states).
- Changes in binding affinities and enantioselectivities were observed with receptor conformational shifts.
- Immobilized P-glycoprotein exhibited ligand-induced allosteric interactions and ATP-driven conformational changes.
- Demonstrated that immobilized proteins maintain conformational mobility.
Conclusions:
- Immobilized receptor and transporter columns retain functional conformational dynamics.
- These columns provide an effective platform for characterizing protein conformations and drug interactions.
- This approach offers an attractive alternative for comprehensive protein characterization in drug discovery.
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