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10:44
Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
Published on: October 21, 2016
Experimental conditions can obscure the second high-affinity site in LeuT
Matthias Quick1, Lei Shi, Britta Zehnpfennig
1Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA.
Nature Structural & Molecular Biology
|January 17, 2012
Summary
Neurotransmitter:Na(+) symporters (NSSs) use two substrate sites for transport. Experimental conditions can block the second site, hiding its role in NSS function.
Area of Science:
- Biochemistry
- Structural Biology
- Neuroscience
Background:
- Neurotransmitter:Na(+) symporters (NSSs) are crucial membrane proteins responsible for neurotransmitter reuptake.
- NSSs function via a Na(+)-dependent symport mechanism, and are targets for psychotropic drugs.
- The crystal structure of LeuT revealed a primary substrate (S1) binding site.
Purpose of the Study:
- To investigate the role of a second substrate (S2) site in NSS function.
- To elucidate the molecular mechanism of Na(+)-substrate symport in NSSs.
- To identify potential artifacts in experimental preparations of NSSs.
Main Methods:
- Computational modeling
- Binding assays
- Flux experiments
- Analysis of detergent-solubilized LeuT structure
Main Results:
- A second substrate (S2) site was identified through computational and experimental approaches.
- The S2 site plays a critical role in the allosteric mechanism of Na(+)-substrate symport.
- The S2 site can be inadvertently blocked during the preparation of detergent-solubilized LeuT.
Conclusions:
- The allosteric interaction between S1 and S2 sites is essential for NSS transport mechanism.
- Crystallographic studies of detergent-solubilized NSSs may obscure the function of the S2 site.
- Careful selection of experimental conditions is necessary to accurately study membrane protein function.
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