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Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
Published on: October 9, 2021
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Tryptophan-based fluorophores for studying protein conformational changes.
Poulami Talukder1, Shengxi Chen1, C Tony Liu2
1Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
Bioorganic & Medicinal Chemistry
|October 7, 2014
Summary
New unnatural amino acid fluorescence probes, structurally related to tryptophan, can be incorporated into proteins. These probes allow for tracking protein conformational changes with minimal disruption to enzyme function.
Area of Science:
- Biochemistry
- Biophysics
- Molecular Biology
Background:
- Understanding protein conformational changes is crucial for deciphering protein function.
- Small fluorescence probes are valuable tools for investigating the crowded interior of proteins.
- Tryptophan fluorescence is a common method for studying protein dynamics, but its spectral properties can be limiting.
Purpose of the Study:
- To introduce and characterize novel unnatural amino acid fluorescence donors as probes for protein conformational changes.
- To assess the compatibility and functional impact of these probes within a protein system.
- To demonstrate the utility of these probes in studying protein dynamics using Förster Resonance Energy Transfer (FRET).
Main Methods:
- Synthesis and characterization of six unnatural amino acid fluorescence donors structurally related to tryptophan.
- Incorporation of these amino acids into Escherichia coli dihydrofolate reductase (DHFR) via genetic methods.
- Photophysical characterization of the incorporated probes, including fluorescence spectra and FRET efficiency.
- Assessment of enzyme activity of engineered DHFR variants.
Main Results:
- The novel tryptophan analogues exhibit redshifted fluorescence spectra compared to native tryptophan.
- Most analogues were well tolerated in DHFR, with minimal impact on enzyme activity.
- Specific substitutions demonstrated efficient Förster Resonance Energy Transfer (FRET) with acridon-2-ylalanine (Acd).
- The probes enabled selective study of conformational changes even in the presence of native tryptophans.
Conclusions:
- Unnatural tryptophan analogues can be efficiently incorporated into proteins like DHFR with minimal functional disruption.
- These novel probes offer redshifted fluorescence properties and can be utilized for FRET-based studies.
- The developed probes are suitable for selectively investigating protein conformational dynamics in complex biological environments.
