Journal
CHEMICAL SCIENCE
Volume 8, Issue 6, Pages 4546-4557Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c7sc00855d
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Funding
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]
- US Federal funds from the National Cancer Institute of the National Institutes of Health (NIH) [HHSN261200800001E]
- NIH [GM068552, GM122567, NS099573]
- EU FP7 [ERC-2013-ADG-340233]
- Russian Science Foundation [17-74-20100]
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Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties. We have identified the residues responsible for the spectral red-shift, revealed a new chromophore bound simultaneously to two cysteine residues in the PAS and GAF domains in blue-shifted NIR FPs, and uncovered the importance of amino acid residues in the N-terminus of NIR FPs for their molecular and cellular brightness. The novel chromophore covalently links the N-terminus of NIR FPs with their C-terminal GAF domain, forming a topologically closed knot in the structure, and also contributes to the increased brightness. Based on our studies, we suggest a strategy to develop spectrally distinct NIR FPs with enhanced brightness.
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