Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.

ABSTRACT: Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R) on PKA's catalytic subunit. We discover tha

SUBMITTER: Lee SR 

PROVIDER: S-EPMC4814300 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature