Dataset Information

A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.

ABSTRACT: Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400% in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far.

SUBMITTER: Mank M

PROVIDER: S-EPMC1367327 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.

Mank Marco M Reiff Dierk F DF Heim Nicola N Friedrich Michael W MW Borst Alexander A Griesbeck Oliver O

Biophysical journal 20051209 5

Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within th ...[more]

PMID: 16339891

Similar Datasets

Project description:We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer (FRET) spectroscopy for probing conformational dynamics of a fast-folding protein, α3D. In three-color FRET, site-specific labeling of fluorophores is required to avoid ambiguity resulting from various species with different combinations of labeling positions. To this end, we first attached two dyes to a cysteine residue and an unnatural amino acid and then appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for attaching the third dye. To determine all three FRET efficiencies, we used alternating excitation of the donor and acceptor 1 with two picosecond-pulsed lasers. Since the folded and unfolded states are not distinguishable in binned fluorescence trajectories due to fast-folding on a millisecond time scale, we used a maximum likelihood method that analyzes photon trajectories without binning the data. The extracted kinetic parameters agree very well with the previously measured parameters for the same protein with two-color FRET, suggesting that the addition of the third fluorophore does not affect the folding dynamics of the protein. From the extracted fractions of acceptor photon counts, the FRET efficiencies for all three dye pairs were calculated after various corrections. They were compared with the FRET efficiencies obtained from the global analysis of two-color segments collected in the same experiment. The FRET efficiencies of the folded state from the three-color segments agree with those from the two-color segments, whereas the three-color and two-color FRET efficiencies of the unfolded state are different. This happens because fluctuations of all three interdye distances contribute to the FRET efficiency measured in three-color FRET. We show that this difference can be accounted for by using the Gaussian chain model for the unfolded state with the parameters obtained from the analysis of two-color segments. This result shows that three-color FRET provides additional information on the flexibility of molecules that cannot be obtained from a combination of two-color FRET experiments with three dye pairs. Using the delay times of photons from the laser pulse, fluorescence lifetimes were determined using the maximum likelihood analysis. The correlation between FRET efficiencies and lifetimes of the donor, acceptor 1, and acceptor 2 was visualized in two-dimensional FRET efficiency-lifetime histograms. These histograms can be used to demonstrate the presence of conformational dynamics in a protein.

Dataset Information

A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.

Publications

A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets