Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein
Lindquist, Jake R.
Davidson, Michael W.
Hughes, Thomas E.
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CitationGuan, Y., M. Meurer, S. Raghavan, A. Rebane, J. R. Lindquist, S. Santos, I. Kats, et al. 2015. “Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.” Molecular Biology of the Cell 26 (11): 2054-2066. doi:10.1091/mbc.E14-10-1473. http://dx.doi.org/10.1091/mbc.E14-10-1473.
AbstractWe 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.
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