Dataset Information

A dansyl fluorescence-based assay for monitoring kinetics of lipid extraction and transfer.

ABSTRACT: Lipid transfer proteins have important roles in cellular biology, and fluorescence spectroscopy has found wide range use as a facile means for time-resolved monitoring of protein-lipid interactions. Here, we show how the fluorescence emission properties of dansyl-DHPE can be exploited to characterize lipid extraction and lipid transfer kinetics. The GM2 activator protein serves as an example of a lipid transfer protein where the ability to independently characterize lipid extraction from donor vesicles, formation of a protein:lipid complex in solution, and release of lipid from the complex to acceptor liposomes is crucial for full kinetic characterization of lipid transfer.

SUBMITTER: Ran Y

PROVIDER: S-EPMC2633774 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A dansyl fluorescence-based assay for monitoring kinetics of lipid extraction and transfer.

Ran Yong Y Fanucci Gail E GE

Analytical biochemistry 20080729 2

Lipid transfer proteins have important roles in cellular biology, and fluorescence spectroscopy has found wide range use as a facile means for time-resolved monitoring of protein-lipid interactions. Here, we show how the fluorescence emission properties of dansyl-DHPE can be exploited to characterize lipid extraction and lipid transfer kinetics. The GM2 activator protein serves as an example of a lipid transfer protein where the ability to independently characterize lipid extraction from donor v ...[more]

PMID: 18694718

Similar Datasets

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

A dansyl fluorescence-based assay for monitoring kinetics of lipid extraction and transfer.

Publications

A dansyl fluorescence-based assay for monitoring kinetics of lipid extraction and transfer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets