Dataset Information

Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.

ABSTRACT: The discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) and the development of genetically encoded fluorescent biosensors. These fluorescence protein-based sensors are highly effective in cells grown in monolayer cultures. However, it is often desirable to use more complex models including tissue explants, organoids, xenografts, and whole animals. These types of samples have poor light penetration owing to high scattering and absorption of light by tissue. Far-red light with a wavelength between 650-900nm is less prone to scatter, and absorption by tissues and can thus penetrate more deeply. Unfortunately, there are few fluorescent proteins in this region of the spectrum, and they have sub-optimal fluorescent properties including low brightness and short fluorescence lifetimes. Understanding the relationships between the amino-acid sequences of far-red fluorescence proteins and their photophysical properties including peak emission wavelengths and fluorescence lifetimes would be useful in the design of new fluorescence proteins for this region of the spectrum. We used both site-directed mutagenesis and gene-shuffling between mScarlet and mCardinal fluorescence proteins to create new variants and assess their properties systematically. We discovered that for far-red, GFP-like proteins the emission maxima and fluorescence lifetime have a strong inverse correlation.

SUBMITTER: Canty L

PROVIDER: S-EPMC6261627 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.

Canty Laura L Hariharan Santosh S Liu Qian Q Haney Steven A SA Andrews David W DW

PloS one 20181128 11

The discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) and the development of genetically encoded fluorescent biosensors. These fluorescence protein-based sensors are highly effective in cells grown in monolay ...[more]

PMID: 30485364

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Project description:Genetically encoded probes with red-shifted absorption and fluorescence are highly desirable for imaging applications because they can report from deeper tissue layers with lower background and because they provide additional colors for multicolor imaging. Unfortunately, red and especially far-red fluorescent proteins have very low quantum yields, which undermines their other advantages. Elucidating the mechanism of nonradiative relaxation in red fluorescent proteins (RFPs) could help developing ones with higher quantum yields. Here we consider two possible mechanisms of fast nonradiative relaxation of electronic excitation in RFPs. The first, known as the energy gap law, predicts a steep exponential drop of fluorescence quantum yield with a systematic red shift of fluorescence frequency. In this case the relaxation of excitation occurs in the chromophore without any significant changes of its geometry. The second mechanism is related to a twisted intramolecular charge transfer in the excited state, followed by an ultrafast internal conversion. The chromophore twisting can strongly depend on the local electric field because the field can affect the activation energy. We present a spectroscopic method of evaluating local electric fields experienced by the chromophore in the protein environment. The method is based on linear and two-photon absorption spectroscopy, as well as on quantum-mechanically calculated parameters of the isolated chromophore. Using this method, which is substantiated by our molecular dynamics simulations, we obtain the components of electric field in the chromophore plane for seven different RFPs with the same chromophore structure. We find that in five of these RFPs, the nonradiative relaxation rate increases with the strength of the field along the chromophore axis directed from the center of imidazolinone ring to the center of phenolate ring. Furthermore, this rate depends on the corresponding electrostatic energy change (calculated from the known fields and charge displacements), in quantitative agreement with the Marcus theory of charge transfer. This result supports the dominant role of the twisted intramolecular charge transfer mechanism over the energy gap law for most of the studied RFPs. It provides important guidelines of how to shift the absorption wavelength of an RFP to the red, while keeping its brightness reasonably high.

Dataset Information

Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.

Publications

Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.

Similar Datasets

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