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Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

ABSTRACT: FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different "Nabi1" constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz.

SUBMITTER: Sung U

PROVIDER: S-EPMC4654489 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

Sung Uhna U Sepehri-Rad Masoud M Piao Hong Hua HH Jin Lei L Hughes Thomas T Cohen Lawrence B LB Baker Bradley J BJ

PloS one 20151120 11

FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphata ...[more]

PMID: 26587834

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Project description:The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f(D)) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f(D) percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f(D) value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f(D) (mf(D)) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f(D) analysis.

Dataset Information

Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

Publications

Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

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