TY - JOUR
T1 - Conversion of Red Fluorescent Protein into a Bright Blue Probe
AU - Subach, Oksana M.
AU - Gundorov, Illia S.
AU - Yoshimura, Masami
AU - Subach, Fedor V.
AU - Zhang, Jinghang
AU - Grüenwald, David
AU - Souslova, Ekaterina A.
AU - Chudakov, Dmitriy M.
AU - Verkhusha, Vladislav V.
N1 - Funding Information:
We thank R. Campbell, P. Daugherty, and M. Davidson for the plasmids with EBFP2 and Azurite genes. We are grateful to W. King for the assistance with flow cytometry. This work was supported by National Institutes of Health grants GM070358 and GM073913 (to V.V.V.) and AA013148 (to M.Y.). Additional support was provided by grants EC FP-6 Integrated Project LSHG-CT-2003-503259 and NATO Collaborative Linkage grant CBP.NR.NRCLG 981752. D.M.C. was supported by grants from the President of the Russian Federation and Rosnauka 02.512.11.2216. D.G. was supported by postdoctoral fellowship GR3388/1 from the Deutsche Forschungsgemeinschaft. The Einstein Flow Cytometry Core Facility was supported by a Cancer Center grant from NCI (CA13330) and by a Center for AIDS Research grant from NIAID (AI051519).
PY - 2008/10/20
Y1 - 2008/10/20
N2 - We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.
AB - We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.
KW - CHEMBIO
UR - http://www.scopus.com/inward/record.url?scp=53649100738&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=53649100738&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2008.08.006
DO - 10.1016/j.chembiol.2008.08.006
M3 - Article
C2 - 18940671
AN - SCOPUS:53649100738
SN - 1074-5521
VL - 15
SP - 1116
EP - 1124
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 10
ER -