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Analysis of protein-ligand interactions by fluorescence polarization.

ABSTRACT: Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a nondisruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP(3)) to N-terminal fragments of IP(3) receptors can be characterized at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (?G°), enthalpy (?H°) and entropy (?S°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real time without the use of radioactive materials, it is nondestructive and, with appropriate care, it can resolve ?H° and ?S°. The first part of the protocol, protein preparation, may take several weeks, whereas the FP measurements, once they have been optimized, would normally take 1-6 h.

SUBMITTER: Rossi AM

PROVIDER: S-EPMC3160472 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Analysis of protein-ligand interactions by fluorescence polarization.

Rossi Ana M AM Taylor Colin W CW

Nature protocols 20110303 3

Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a nondisruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP(3)) to N-terminal fragments of IP(3) receptors can be characterized at different temperatures and in competit ...[more]

PMID: 21372817

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Project description:Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically "dead" BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development. MOTIVATION In vitro BAX activation studies are invaluable platforms for studying cellular and pharmacological modulators of apoptosis. The gold standard for studying BAX function relies on membrane permeabilization assays, which assess the pore-forming activity of oligomeric BAX. However, there are currently no rapid or kinetic assays to interrogate real-time activation of monomeric BAX in solution, thereby limiting any molecular insights that occur upstream of mitochondrial permeabilization. Furthermore, available methods to observe the activation of monomeric BAX suffer from low throughput and static observations. To address this methodological gap, we developed FLAMBE, a kinetic fluorescence polarization-based assay to measure monomeric BAX activation in solution via concomitant displacement of a labeled peptide. This approach maintains the benefits of rapid kinetic data generation in a low-cost microplate format without requiring specialized equipment or large quantities of protein. FLAMBE compliments available experimental strategies and expands the accessibility of investigators to monitor early steps within the BAX activation continuum.

Dataset Information

Analysis of protein-ligand interactions by fluorescence polarization.

Publications

Analysis of protein-ligand interactions by fluorescence polarization.

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