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Information about Fluorescence Resonance Energy Transfer

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Fluorescent proteins localize the guanosine 5'-triphosphate hydrolase ARF in the Golgi apparatus of a living macrophage. FRET studies revealed ARF activation in the Golgi and in the formation of phagosomes.[1]


Fluorescence resonance energy transfer (FRET) describes an energy transfer mechanism between two chromophores.

A donor chromophore in its excited state can transfer energy by a nonradiative, long-range dipole-dipole coupling mechanism to an acceptor chromophore in close proximity (typically <10nm). This energy transfer mechanism is termed "Förster resonance energy transfer" (FRET), named after the German scientist Theodor Förster. When both molecules are fluorescent, the term "fluorescence resonance energy transfer" is often used, although the energy is not actually transferred by fluorescence.[2],[3]. In order to avoid an erroneous interpretation of the phenomenon that, even when occurring between two fluorescent molecules, is always a nonradiative transfer of energy, the name "Förster resonance energy transfer" may be preferred to "Fluorescence resonance energy transfer".

Theoretical basis

The FRET efficiency () is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event:



where is the rate of energy transfer, the radiative decay rate and the are the rate constants of any other de-excitation pathway.

The FRET efficiency depends on many parameters that can be grouped as follows:
  • The distance between the donor and the acceptor
  • The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum.
  • The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.
depends on the donor-to-acceptor separation distance with an inverse 6th power law due to the dipole-dipole coupling mechanism:
with being the Förster distance of this pair of donor and acceptor at which the FRET efficiency is 50%. The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation:
where is the dipole orientation factor, is the refractive index of the medium, is the fluorescence quantum yield of the donor in the absence of the acceptor, and is the spectral overlap integral calculated as
where is the normalized donor emission spectrum, and is the acceptor molar extinction coefficient. κ2 =2/3 is often assumed. This value is obtained when both dyes are freely rotating and can be considered to be isotropically oriented during the excited state lifetime. If either dye is fixed or not free to rotate, then κ2 =2/3 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging that κ2 = 2/3 does not result in a large error in the estimated energy transfer distance due to the sixth power dependence of R0 on κ2. Even when κ2 is quite different from 2/3 the error can be associated with a shift in R0 and thus determinations of changes in relative distance for a particular system are still valid. Fluorescent proteins do not reorient on a timescale that is faster than their fluorescence lifetime. In this case 0 ≤ κ2 ≤ 4.

The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the donor molecule as follows:
where and are the donor fluorescence lifetimes in the presence and absence of an acceptor, respectively, or as
where and are the donor fluorescence intensities with and without an acceptor, respectively.

Methods

In fluorescence microscopy, fluorescence confocal laser scanning microscopy, as well as in molecular biology, FRET is a useful tool to quantify molecular dynamics in biophysics and biochemistry, such as protein-protein interactions, protein-DNA interactions, and protein conformational changes. For monitoring the complex formation between two molecules, one of them is labeled with a donor and the other with an acceptor, and these fluorophore-labeled molecules are mixed. When they are dissociated, the donor emission is detected upon the donor excitation. On the other hand, when the donor and acceptor are in proximity (1-10 nm) due to the interaction of the two molecules, the acceptor emission is predominantly observed because of the intermolecular FRET from the donor to the acceptor. For monitoring protein conformational changes, the target protein is labeled with a donor and an acceptor at two loci. When a twist or bend of the protein brings the change in the distance or relative orientation of the donor and acceptor, FRET change is observed. If a molecular interaction or a protein conformational change is dependent on ligand binding, this FRET technique is applicable to fluorescent indicators for the ligand detection.

FRET studies are scalable: the extent of energy transfer is often quantified from the milliliter scale of cuvette-based experiments to the femtoliter scale of microscopy-based experiments. This quantification can be based directly (sensitized emission method) on detecting two emission channels under two different excitation conditions (primarily donor and primarily acceptor). However, for robustness reasons, FRET quantification is most often based on measuring changes in fluorescence intensity or fluorescence lifetime upon changing the experimental conditions (e.g. a microscope image of donor emission is taken with the acceptor being present. The acceptor is then bleached, such that it is incapable of accepting energy transfer and another donor emission image is acquired. A pixel-based quantification using the second equation in the theory section above is then possible.) An alternative way of temporarily deactivating the acceptor is based on its fluorescence saturation. Exploiting polarisation characteristics of light, a FRET quantification is also possible with only a single camera exposure.

CFP-YFP pairs

The most popular FRET pair for biological use is a cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) pair. Both are color variants of green fluorescent protein (GFP). While labeling with organic fluorescent dyes requires troublesome processes of purification, chemical modification, and intracellular injection of a host protein, GFP variants can be easily attached to a host protein by genetic engineering. By virtue of GFP variants, the use of FRET techniques for biological research is becoming more and more popular.

BRET

A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise in the results from direct excitation of the acceptor or to photobleaching. To avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. This technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than CFP to produce an initial photon emission compatible with YFP.

FRET and BRET are also the common tools in the study of biochemical reaction kinetics and molecular motors.

Other methods

A different, but related, mechanism is Dexter Electron Transfer.

An alternative method to detecting protein-protein proximity is BiFC where two halves of a YFP are fused to a protein (Hu, Kerppola et al. 2002). When these two halves meet they form a fluorophore after about 60 s - 1 hr.

Applications

FRET has been applied in an experimental method for the detection of phosgene. In it, phosgene or rather triphosgene as a safe substitute serves as a linker between an acceptor and a donor coumarine (forming urea groups).[4] The presence of phosgene is detected at 5x10-5M with a typical FRET emission at 464 nm.

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FRET application phosgene detection

References

1. ^ Inconspicuous Consumption: Uncovering the Molecular Pathways behind Phagocytosis. Inman M, PLoS Biology Vol. 4/6/2006, e190. doi:10.1371/journal.pbio.0040190
2. ^ Joseph R. Lakowicz, "Principles of Fluorescence Spectroscopy", Plenum Publishing Corporation, 2nd edition (July 1, 1999)
3. ^ FRET microscopy tutorial from Olympus
4. ^ A FRET approach to phosgene detection Hexiang Zhang and Dmitry M. Rudkevich Chem. Commun., 2007, 1238 - 1239, doi:10.1039/b614725a
A chromophore is part (or moiety) of a molecule responsible for its color.

When a molecule absorbs certain wavelengths of visible light and transmits or reflects others, the molecule has a color.
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dipoles (Hellènic: di(s)- = twi- and pòla = pivot, hinge). An electric dipole is a separation of positive and negative charge. The simplest example of this is a pair of electric charges of equal magnitude but opposite sign, separated by some, usually small,
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An element's emission spectrum is the relative intensity of electromagnetic radiation of each frequency it emits when it is heated (or more generally when it is excited).

When the electrons in the element are excited, they jump to higher energy levels.
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A material's absorption spectrum shows the fraction of incident electromagnetic radiation absorbed by the material over a range of frequencies. An absorption spectrum is, in a sense, the opposite of an emission spectrum.
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INTErnational Gamma-Ray Astrophysics Laboratory (INTEGRAL) is detecting some of the most energetic radiation that comes from space. It is the most sensitive gamma ray observatory ever launched.
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The refractive index (or index of refraction) of a medium is a measure for how much the speed of light (or other waves such as sound waves) is reduced inside the medium. For example, typical glass has a refractive index of 1.
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The quantum yield of a radiation-induced process is the number of times that a defined event occurs per photon absorbed by the system. Thus, the quantum yield is a measure of the efficiency with which absorbed light produces some effect.
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The molar extinction coefficient, also known as molar absorptivity, is a measure of how strongly a chemical species at a given wavelength absorbs light at that wavelength.
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Microscopy is any technique for producing visible images of structures or details too small to otherwise be seen by the human eye, using a microscope or other magnification tool. It is often used more specifically as a technique of using a microscope.
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Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images.[1] The key feature of confocal microscopy is its ability to produce in-focus images of thick specimens, a process known as
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Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell,
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Biophysics (also biological physics) is an interdisciplinary science that applies the theories and methods of physics to questions of biology.

Biophysics research today is comprised of a lot of specific biological studies, which don't share a unique identifying
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Biochemistry is the study of the chemical processes in living organisms.[1] The word "biochemistry" comes from the Greek word βιοχημεία biochēmeia, which means "the chemistry of life.
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Proteins are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.
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A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength.
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In physics, chemistry, and biology, intermolecular forces are forces that act between stable molecules or between functional groups of macromolecules. These non-covalent forces, which give rise to bonding energies of less than a few kcal/mol, are generally much weaker than the
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In chemistry, a ligand is an atom, ion, or molecule (see also: functional group) that generally donates one or more of its electrons through a coordinate covalent bond to, or shares its electrons through a covalent bond with, one or more central atoms or ions (these ligands act as
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green fluorescent protein (GFP) is a protein, comprised of 238 amino acids (26,9 kDa), from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light.
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Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that are applied to the direct manipulation of an organisms genes.
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Photobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing.
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Bioluminescence is the production and emission of light by a living organism as the result of a chemical reaction during which chemical energy is converted to light energy.
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Luciferase is a generic name for enzymes commonly used in nature for bioluminescence. The name itself is derived from Lucifer, which means light-bearer. The most famous one is firefly luciferase (EC 1.13.12.7 ) from the firefly Photinus pyralis.
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'Kinetics', derived from the Greek word κίνησις (kinesis) meaning "movement", may refer to:

Science

  • Chemical kinetics, the study of chemical reaction rates.

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Molecular motors are biological molecular machines that are the essential agents of movement in living organisms. Generally speaking, a motor may be defined as a device that consumes energy in one form and converts it into motion or mechanical work; for example, many protein-based
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Bimolecular fluorescence complementation (BiFC) is a method of viewing the association of proteins inside living cells. Kerppola et al. (2002) solidified this methodology as viable in vivo.
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Phosgene is a highly reactive toxic chemical compound with the formula CCl2O.

This gas gained infamy as a chemical weapon during World War I, but it is also a valuable industrial reagent and building block in organic synthesis.
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Triphosgene (Bis(trichloromethyl) carbonate, C3Cl6O3) is a chemical compound that is used as a substitute for phosgene. At room temperature, the compound is a white crystalline solid that decomposes around 130 °C.
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Coumarin is a chemical compound(benzopyrone); a toxin found in many plants, notably in high concentration in the tonka bean, woodruff, and bison grass. It has a sweet scent, readily recognised as the scent of newly-mown hay.
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Urea is an organic compound with the chemical formula (NH2)2CO.

Urea is also known as carbamide, especially in the recommended International Nonproprietary Names (rINN) in use in Europe.
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