TY - JOUR
T1 - Molecular Basis of Spectral Diversity in Near-Infrared Phytochrome-Based Fluorescent Proteins
AU - Shcherbakova, Daria M.
AU - Baloban, Mikhail
AU - Pletnev, Sergei
AU - Malashkevich, Vladimir N.
AU - Xiao, Hui
AU - Dauter, Zbigniew
AU - Verkhusha, Vladislav V.
N1 - Funding Information:
We thank Eric Giraud (Institute for Research and Development, Montpellier, France) for the Rp BphP1 gene, Clark Lagarias (University of California at Davis) and Richard Vierstra (University of Wisconsin at Madison) for the plasmids for production of BV and PΦB in bacteria. We are grateful to Clark Lagarias for useful comments on the manuscript. We thank Jinghang Zhang for assistance with flow cytometry. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. W-31-109-Eng-38 . This work was supported in part with US Federal funds from the National Cancer Institute of the NIH under contract HHSN261200800001E , the Intramural Research Program of the NIH , by the NIH grants CA164468 , GM073913 and GM108579 (all to V.V.V.) and ERC-2013-ADG-340233 (to V.V.V.) grant from the EU FP7 program .
PY - 2015/11/19
Y1 - 2015/11/19
N2 - Summary Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are the probes of choice for deep-tissue imaging. Detection of several processes requires spectrally distinct NIR FPs. We developed an NIR FP, BphP1-FP, which has the most blue-shifted spectra and the highest fluorescence quantum yield among BphP-derived FPs. We found that these properties result from the binding of the biliverdin chromophore to a cysteine residue in the GAF domain, unlike natural BphPs and other BphP-based FPs. To elucidate the molecular basis of the spectral shift, we applied biochemical, structural and mass spectrometry analyses and revealed the formation of unique chromophore species. Mutagenesis of NIR FPs of different origins indicated that the mechanism of the spectral shift is general and can be used to design multicolor NIR FPs from other BphPs. We applied pairs of spectrally distinct point cysteine mutants to multicolor cell labeling and demonstrated that they perform well in model deep-tissue imaging.
AB - Summary Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are the probes of choice for deep-tissue imaging. Detection of several processes requires spectrally distinct NIR FPs. We developed an NIR FP, BphP1-FP, which has the most blue-shifted spectra and the highest fluorescence quantum yield among BphP-derived FPs. We found that these properties result from the binding of the biliverdin chromophore to a cysteine residue in the GAF domain, unlike natural BphPs and other BphP-based FPs. To elucidate the molecular basis of the spectral shift, we applied biochemical, structural and mass spectrometry analyses and revealed the formation of unique chromophore species. Mutagenesis of NIR FPs of different origins indicated that the mechanism of the spectral shift is general and can be used to design multicolor NIR FPs from other BphPs. We applied pairs of spectrally distinct point cysteine mutants to multicolor cell labeling and demonstrated that they perform well in model deep-tissue imaging.
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U2 - 10.1016/j.chembiol.2015.10.007
DO - 10.1016/j.chembiol.2015.10.007
M3 - Article
C2 - 26590639
AN - SCOPUS:84947915622
VL - 22
SP - 1540
EP - 1551
JO - Cell Chemical Biology
JF - Cell Chemical Biology
SN - 2451-9448
IS - 11
ER -