Quantitative measurements of protein interactions in a crowded cellular environment.
Quantitative measurements of protein interaction strengths, crucial for describing signaling networks and predicting cellular responses to environmental stimuli, are typically performed in dilute buffer solutions. However, protein-protein interactions in cells occur within the context of a crowded system, which is characterized by a high macromolecular concentration. In this paper, we explore the utility of cell-derived vesicles as a model crowded environment for quantitative FRET measurements of protein-protein interactions. We show that the FRET efficiency, and the donor and acceptor concentrations, can be calculated in each vesicle. We also introduce the "quantitative imaging Foster resonance energy transfer" method as a tool that can yield protein interaction strengths within these vesicles.
Li
Edwin
E
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Placone
Jesse
J
Merzlyakov
Mikhail
M
Hristova
Kalina
K
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D008962:Models, Theoretical; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014162:Transfection
D049452:Green Fluorescent Proteins