Author: Krause CD, Digioia G, Izotova LS, Pestka S.
Affiliation: Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, The University of Medicine and Dentistry of New Jersey, 675 Hoes Lane West, Piscataway, NJ 08855, USA. Electronic address: christopher.d.krause@gmail.com.
Conference/Journal: Cytokine.
Date published: 2013 Jun 21
Other:
Pages: S1043-4666(13)00272-X , Special Notes: doi: 10.1016/j.cyto.2013.05.026 , Word Count: 186
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Copyright © 2013. Published by Elsevier Ltd.
PMID: 23796694