RESUMO
Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.
Assuntos
Proteínas de Fluorescência Verde/química , Animais , Animais Geneticamente Modificados , Ânions/química , Drosophila , Escherichia coli , Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Mutação , Processos Fotoquímicos , Temperatura , Triptofano/química , Triptofano/metabolismoRESUMO
We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (â¼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (â¼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein-protein interactions. We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.
