Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy.

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.

[1]  S. Lukyanov,et al.  Fluorescent proteins from nonbioluminescent Anthozoa species , 1999, Nature Biotechnology.

[2]  G Ulrich Nienhaus,et al.  A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria) , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  S J Remington,et al.  Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[4]  O. V. Stepanenko,et al.  Comparative studies on the structure and stability of fluorescent proteins EGFP, zFP506, mRFP1, "dimer2", and DsRed1. , 2004, Biochemistry.

[5]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  James H. Werner,et al.  Single molecule spectroscopic characterization of a far-red fluorescent protein (HcRed) from the Anthozoa coral Heteractis crispa , 2006, SPIE BiOS.

[7]  Mark Van der Auweraer,et al.  New picosecond laser system for easy tunability over the whole ultraviolet/visible/near infrared wavelength range based on flexible harmonic generation and optical parametric oscillation , 2001 .

[8]  M. J. Cormier,et al.  Primary structure of the Aequorea victoria green-fluorescent protein. , 1992, Gene.

[9]  J Michiels,et al.  Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Gregor Jung,et al.  The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222. , 2005, Biophysical journal.

[11]  Joachim Goedhart,et al.  Bright monomeric red fluorescent protein with an extended fluorescence lifetime , 2007, Nature Methods.

[12]  J. Enderlein,et al.  Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration. , 2005, Chemphyschem : a European journal of chemical physics and physical chemistry.

[13]  N. F. Hulst,et al.  The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Guido Jach,et al.  An improved mRFP1 adds red to bimolecular fluorescence complementation , 2006, Nature Methods.

[15]  Thomas Dertinger,et al.  Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. , 2007, Chemphyschem : a European journal of chemical physics and physical chemistry.

[16]  G Ulrich Nienhaus,et al.  Photodynamics of red fluorescent proteins studied by fluorescence correlation spectroscopy. , 2004, Biophysical journal.

[17]  Sean C. Smith,et al.  A structural basis for the pH-dependent increase in fluorescence efficiency of chromoproteins. , 2007, Journal of molecular biology.

[18]  Vladislav V Verkhusha,et al.  Conversion of the monomeric red fluorescent protein into a photoactivatable probe. , 2005, Chemistry & biology.

[19]  J. Antosiewicz,et al.  Empirical relationships between protein structure and carboxyl pKa values in proteins , 2002, Proteins.

[20]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[21]  W E Moerner,et al.  Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[22]  O. Shimomura,et al.  The discovery of aequorin and green fluorescent protein , 2005, Journal of microscopy.

[23]  Jerker Widengren,et al.  Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy , 1999 .

[24]  S. Brooks.  The discovery of aequorin and green fluorescent protein , 2005 .

[25]  Mircea Cotlet,et al.  Collective effects in individual oligomers of the red fluorescent coral protein DsRed , 2001 .

[26]  G. Mocz.  Fluorescent Proteins and Their Use in Marine Biosciences, Biotechnology, and Proteomics , 2007, Marine Biotechnology.

[27]  G. Orlovsky,et al.  The kindling fluorescent protein: a transient photoswitchable marker. , 2006, Physiology.

[28]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[29]  R. Tsien,et al.  Evolution of new nonantibody proteins via iterative somatic hypermutation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Jelle Hendrix,et al.  Overexpression of the Lens Epithelium-Derived Growth Factor/p75 Integrase Binding Domain Inhibits Human Immunodeficiency Virus Replication , 2006, Journal of Virology.

[31]  P. Schwille,et al.  Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states. , 2001, Biophysical journal.

[32]  O. Hoegh‐Guldberg,et al.  The 2.2 A crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. , 2003, Structure.

[33]  Kenneth D. Weston,et al.  Probing Förster Type Energy Pathways in a First Generation Rigid Dendrimer Bearing Two Perylene Imide Chromophores , 2003 .

[34]  J. Hofkens,et al.  Photoconversion in the red fluorescent protein from the sea anemone Entacmaea quadricolor: is cis-trans isomerization involved? , 2006, Journal of the American Chemical Society.

[35]  J. Hofkens,et al.  Singlet oxygen photosensitization by EGFP and its chromophore HBDI. , 2008, Biophysical journal.

[36]  Nathan C Shaner,et al.  Novel chromophores and buried charges control color in mFruits. , 2006, Biochemistry.

[37]  J A McCammon,et al.  Shedding light on the dark and weakly fluorescent states of green fluorescent proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[38]  W. Webb,et al.  Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[39]  S T Hess,et al.  Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Michiels,et al.  Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed. , 2005, Journal of the American Chemical Society.

[41]  K K Baldridge,et al.  The structure of the chromophore within DsRed, a red fluorescent protein from coral. , 2000, Proceedings of the National Academy of Sciences of the United States of America.