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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
Published on: March 11, 2021
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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time.
Samuel G Usher1, Frances M Ashcroft2, Michael C Puljung3
1Department of Physiology, Anatomy and Genetics, University of Oxford.
Journal of Visualized Experiments : Jove
|March 29, 2021
Summary
We developed a new method to measure adenine nucleotide binding to transmembrane receptors using fluorescent tags. This technique offers insights into protein function and can be adapted for various nucleotide-regulated proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Adenine nucleotides regulate numerous cellular processes.
- Understanding nucleotide-receptor interactions is crucial for drug development.
- Existing methods for studying these interactions in native environments are limited.
Purpose of the Study:
- To develop a novel method for measuring adenine nucleotide binding to intact transmembrane receptors.
- To characterize nucleotide binding to KATP ion channels.
- To provide a versatile tool for studying nucleotide-regulated proteins.
Main Methods:
- Utilized ANAP (a fluorescent non-canonical amino acid) tagging of proteins.
- Employed Förster Resonance Energy Transfer (FRET) between ANAP and fluorescent nucleotide derivatives.
- Measured binding in cellular or membrane environments, including excised membrane patches under voltage clamp.
Main Results:
- Successfully measured adenine nucleotide binding to ANAP-tagged KATP ion channels.
- Demonstrated simultaneous measurement of ligand binding and channel current.
- Validated the method's ability to provide mechanistic insights into ligand-dependent gating.
Conclusions:
- The developed FRET-based method enables quantitative measurement of nucleotide binding to functional receptors.
- This technique offers significant advantages for studying ligand-dependent protein regulation.
- The method is adaptable to a wide range of nucleotide-binding proteins and receptors.
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