Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein


Yinghua Guan, Matthias Meurer, Sarada Raghavan, Aleksander Rebane, Jake R. Lindquist, Sofia Santos, Ilia Kats, Michael W. Davidson, Ralph Mazitschek, Thomas E. Hughes, Mikhail Drobizhev, Michael Knop, Jagesh V. Shah


Molecular Biology of the Cell


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.



How is this information collected?

This collection of Montana State authored publications is collected by the Library to highlight the achievements of Montana State researchers and more fully understand the research output of the University. They use a number of resources to pull together as complete a list as possible and understand that there may be publications that are missed. If you note the omission of a current publication or want to know more about the collection and display of this information email Leila Sterman.