Format

Send to:

Choose Destination
See comment in PubMed Commons below
PLoS One. 2012;7(11):e49593. doi: 10.1371/journal.pone.0049593. Epub 2012 Nov 13.

The impact of heterogeneity and dark acceptor states on FRET: implications for using fluorescent protein donors and acceptors.

Author information

  • 1Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, United States of America. stevevog@mail.nih.gov

Erratum in

  • PLoS One. 2013;8(1). doi:10.1371/annotation/ea42b98e-12b7-4940-8c4e-6142dedbb997.

Abstract

Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.

PMID:
23152925
[PubMed - indexed for MEDLINE]
PMCID:
PMC3496711
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Public Library of Science Icon for PubMed Central
    Loading ...
    Write to the Help Desk