SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Dark States in Monomeric Red Fluorescent Proteins studied by Fluorescence Correlation and Single Molecule Spectroscopy
Jelle Hendrix 1, Cristina Flors 1, Peter Dedecker 1, Johan Hofkens 1 and Yves Engelborghs 2*
1 Katholieke Universiteit Leuven
2 K.U. Leuven
* To whom correspondence should be addressed. E-mail: yves.engelborghs{at}fys.kuleuven.be.
Submitted on October 9, 2007
Revised on November 6, 2007
Accepted on 21 December 2007
 |
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 (FCS) 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-colour FCS 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.
Key Words:
Flickering, H-bond, Triplet, cis-trans isomerization, diffusion, microscopy
