Title: Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy
Authors: Hendrix, Jelle ×
Flors, Cristina
Dedecker, Peter
Hofkens, Johan
Engelborghs, Yves #
Issue Date: May-2008
Publisher: Biophysical Society
Series Title: Biophysical Journal vol:94 issue:10 pages:4103-4113
Abstract: Monomeric red fluorescent proteins (mRFPs) have become indispensable tools for studying protein dynamics, interactions and functions in the cellular environment. Their emission spectrum can be well separated from other fluorescent proteins, and their monomeric structure preserves the natural function of fusion proteins. However, previous photophysical studies of some RFPs have shown the presence of light-induced dark states that can complicate the interpretation of cellular experiments. In this article, we extend these studies to mRFP1, mCherry, and mStrawberry by means of fluorescence correlation spectroscopy and prove that this light-driven intensity flickering also occurs in these proteins. Furthermore, we show that the flickering in these proteins is pH-dependent. Single molecule spectroscopy revealed reversible transitions from a bright to a dark state in several timescales, even up to seconds. Time-resolved fluorescence spectroscopy showed multiexponential decays, consistent with a "loose" conformation. We offer a structural basis for the fluorescence flickering using known crystal structures and point out that the environment of Glu-215 is critical for the pH dependence of the flickering in RFPs. We apply dual-color fluorescence correlation spectroscopy inside live cells to prove that this flickering can seriously hamper cellular measurements if the timescales of the flickering and diffusion are not well separated.
ISSN: 0006-3495
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Biochemistry, Molecular and Structural Biology Section
Molecular Imaging and Photonics
× corresponding author
# (joint) last author

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