mBeRFP, an Improved Large Stokes Shift Red Fluorescent Protein

Herein, we describe the generation of a monomeric large Stokes shift (LSS) red fluorescent protein, mBeRFP, with excitation and emission peaks at 446 and 615 nm, respectively. Compared with two previously reported LSS-RFPs (mKeima and LSS-mKate2), mBeRFP is approximately three times brighter. In addition, mBeRFP is characterized by improved photostability, rapid maturation, an extended lifetime, and a monomeric nature. Additionally, mBeRFP can be paired with the Alexa 647 dye as a FRET donor to detect caspase 3 activity. This FRET pair has an extremely dynamic range and a large Förster radius (approximately 6.5 nm). To demonstrate the applicability of mBeRFP for imaging in living cells, we performed dual-color imaging of mBeRFP and CFP simultaneously excited by a single excitation source, and we demonstrated that these fluorescent proteins allow the clear visualization of the dynamics of Bax during cancer cell apoptosis. Thus, mBeRFP appears to be particularly useful for cellular imaging applications.

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

[2]  A. Miyawaki,et al.  Two-photon dual-color imaging using fluorescent proteins , 2008, Nature Methods.

[3]  Atsushi Miyawaki,et al.  A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy , 2006, Nature Biotechnology.

[4]  W. Zipfel,et al.  Optimization of Pairings and Detection Conditions for Measurement of FRET between Cyan and Yellow Fluorescent Proteins , 2006, Microscopy and Microanalysis.

[5]  A. Mayevsky.  MITOCHONDRIAL FUNCTION AND TISSUE VIABILITY IN VIVO: FROM ANIMAL EXPERIMENTS TO CLINICAL APPLICATIONS. FORTY YEARS OF FRUITFUL COLLABORATION WITH BRITTON CHANCE , 2011 .

[6]  G. Ulrich Nienhaus,et al.  mRuby, a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellular Structures , 2009, PloS one.

[7]  S. Frank,et al.  Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis , 2002, The Journal of cell biology.

[8]  Robert E Campbell,et al.  Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors , 2008, Nature Methods.

[9]  S. D. Varfolomeyev,et al.  Kinetic Analysis of Maturation and Denaturation of DsRed, a Coral-Derived Red Fluorescent Protein , 2001, Biochemistry (Moscow).

[10]  Konstantin A Lukyanov,et al.  Fluorescent proteins as a toolkit for in vivo imaging. , 2005, Trends in biotechnology.

[11]  Joachim Goedhart,et al.  Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples , 2007, PloS one.

[12]  Liisa M. Hirvonen,et al.  DEEP-UV CONFOCAL FLUORESCENCE IMAGING AND SUPER-RESOLUTION OPTICAL MICROSCOPY OF BIOLOGICAL SAMPLES , 2012 .

[13]  Oliver Griesbeck,et al.  Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP , 2003, BMC biotechnology.

[14]  G. Patterson.  A new harvest of fluorescent proteins , 2004, Nature Biotechnology.

[15]  Vladislav V Verkhusha,et al.  An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. , 2012, Journal of the American Chemical Society.

[16]  R. W. Draft,et al.  Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system , 2007, Nature.

[17]  Bin Wu,et al.  Monomeric red fluorescent proteins with a large Stokes shift , 2010, Proceedings of the National Academy of Sciences.

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

[19]  Zbigniew Dauter,et al.  A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore* , 2008, Journal of Biological Chemistry.

[20]  Shaoqun Zeng,et al.  A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions. , 2009, Biosensors & bioelectronics.

[21]  M. Davidson,et al.  Advances in fluorescent protein technology , 2011, Journal of Cell Science.

[22]  D. Shcherbo,et al.  Bright far-red fluorescent protein for whole-body imaging , 2007, Nature Methods.

[23]  N. Tinel,et al.  Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization , 2008, Nature Methods.

[24]  Mark A Rizzo,et al.  An improved cyan fluorescent protein variant useful for FRET , 2004, Nature Biotechnology.