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

Mol Biol Cell
Authors
Keywords
Abstract

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.

Year of Publication
2015
Journal
Mol Biol Cell
Volume
26
Issue
11
Pages
2054-66
Date Published
2015 Jun 01
ISSN
1939-4586
DOI
10.1091/mbc.E14-10-1473
PubMed ID
25877871
PubMed Central ID
PMC4472016
Links
Grant list
NS083875 / NS / NINDS NIH HHS / United States
GM098083 / GM / NIGMS NIH HHS / United States
R01 NS083875 / NS / NINDS NIH HHS / United States
GM77238 / GM / NIGMS NIH HHS / United States
R01 GM077238 / GM / NIGMS NIH HHS / United States
R01 GM098083 / GM / NIGMS NIH HHS / United States