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

Guan Y, Meurer M, Raghavan S, Rebane A, Lindquist JR, Santos S, Kats I, Davidson MW, Mazitschek R, Hughes TE, Drobizhev M, Knop M, Shah JV - Mol. Biol. Cell (2015)

Bottom Line: We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.

No MeSH data available.


Related in: MedlinePlus

Evolved variants of the long Stokes shift fluorescent protein mKeima. (A) Alignment of mKeima variants. (B) Normalized absorption (dashed lines) and fluorescence emission (solid lines) spectra of hmKeima8.5 (dark blue/red) and mTFP1 (blue/teal). (C) The two-photon cross sections of mTFP1 and hmKeima8.5.
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Figure 1: Evolved variants of the long Stokes shift fluorescent protein mKeima. (A) Alignment of mKeima variants. (B) Normalized absorption (dashed lines) and fluorescence emission (solid lines) spectra of hmKeima8.5 (dark blue/red) and mTFP1 (blue/teal). (C) The two-photon cross sections of mTFP1 and hmKeima8.5.

Mentions: DNA shuffling (Crameri et al., 1996) was used to evolve improved mKeima mutants using budding yeast as a host organism (see Materials and Methods). Two brighter variants were chosen for further characterization, mKeima4.15 (from round 4) and mKeima8.5 (from round 8) (Figure 1A).


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

Guan Y, Meurer M, Raghavan S, Rebane A, Lindquist JR, Santos S, Kats I, Davidson MW, Mazitschek R, Hughes TE, Drobizhev M, Knop M, Shah JV - Mol. Biol. Cell (2015)

Evolved variants of the long Stokes shift fluorescent protein mKeima. (A) Alignment of mKeima variants. (B) Normalized absorption (dashed lines) and fluorescence emission (solid lines) spectra of hmKeima8.5 (dark blue/red) and mTFP1 (blue/teal). (C) The two-photon cross sections of mTFP1 and hmKeima8.5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4472016&req=5

Figure 1: Evolved variants of the long Stokes shift fluorescent protein mKeima. (A) Alignment of mKeima variants. (B) Normalized absorption (dashed lines) and fluorescence emission (solid lines) spectra of hmKeima8.5 (dark blue/red) and mTFP1 (blue/teal). (C) The two-photon cross sections of mTFP1 and hmKeima8.5.
Mentions: DNA shuffling (Crameri et al., 1996) was used to evolve improved mKeima mutants using budding yeast as a host organism (see Materials and Methods). Two brighter variants were chosen for further characterization, mKeima4.15 (from round 4) and mKeima8.5 (from round 8) (Figure 1A).

Bottom Line: We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.

No MeSH data available.


Related in: MedlinePlus