Guan Yinghua, Meurer Matthias, Raghavan Sarada, Rebane Aleksander, Lindquist Jake R, Santos Sofia, Kats Ilia, Davidson Michael W, Mazitschek Ralph, Hughes Thomas E, Drobizhev Mikhail, Knop Michael, Shah Jagesh V
Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.
Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany.
Mol Biol Cell. 2015 Jun 1;26(11):2054-66. doi: 10.1091/mbc.E14-10-1473. Epub 2015 Apr 15.
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 (∼180 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 (∼120-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.
我们报道了一种单体长斯托克斯位移红色荧光蛋白mKeima的改进变体hmKeima8.5。其细胞内亮度的增加和大斯托克斯位移（约180纳米）使其成为用于多光子、多色应用的与蓝绿色荧光蛋白（mTFP1）的绝佳搭档。通过单一多光子激发波长（MPE，850纳米）激发这一对荧光蛋白会产生可很好分离的发射峰（约120纳米的间隔）。利用这一对荧光蛋白，我们通过多光子激发荧光相关光谱法（MPE-FCS）测量活细胞中的同型和异型寡聚化相互作用。使用串联二聚体蛋白和小分子诱导二聚化结构域，我们展示了对细胞内蛋白质-蛋白质相互作用的强大且定量的检测。我们还使用MPE-FCCS，利用香豆素343（C343）偶联药物和hmKeima8.5作为荧光对来检测细胞内环境中的药物-蛋白质相互作用。mTFP1/hmKeima8.5和C343/hmKeima8.5组合，连同我们的校准构建体，为研究活细胞细胞质中的分子相互作用提供了一个实用且广泛适用的工具箱。
