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. Image File history File links Size of this preview: 600 × 600 pixelsFull resolution (750 × 750 pixel, file size: 63 KB, MIME type: image/png) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Fluorescence resonance energy transfer ... Image File history File links Size of this preview: 600 × 600 pixelsFull resolution (750 × 750 pixel, file size: 63 KB, MIME type: image/png) File links The following pages on the English Wikipedia link to this file (pages on other projects are not listed): Fluorescence resonance energy transfer ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... The chemical structure of Guanosine Guanosine is a nucleoside comprising guanine attached to a ribose (ribofuranose) ring via a β-N9-glycosidic bond. ... Guanosine triphosphate (GTP) is also known as guanosine-5-triphosphate. ... In biochemistry, a hydrolase is an enzyme that can break a chemical bond by hydrolysis. ... Micrograph of Golgi apparatus, visible as a stack of semicircular black rings near the bottom. ... A macrophage of a mouse stretching its arms to engulf two particles, possibly pathogens Macrophages (Greek: big eaters, makros = long, phagein = eat) are white blood cells, more specifically phagocytes, acting in the nonspecific defense as well as the specific defense system of vertebrate animals. ... A chromophore is part (or moiety) of a molecule responsible for its color. ...

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". The Earths magnetic field, which is approximately a dipole. ...

Theoretical basis

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

where k_ET is the rate of energy transfer, k_f the radiative decay rate and the k_i 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.

E depends on the donor-to-acceptor separation distance r with an inverse 6th power law due to the dipole-dipole coupling mechanism: An elements 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). ... A materials absorption spectrum shows the fraction of incident electromagnetic radiation absorbed by the material over a range of frequencies. ...

with R₀ 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: This article is about the concept of integrals in calculus. ...

where κ² is the dipole orientation factor, n is the refractive index of the medium, Q₀ is the fluorescence quantum yield of the donor in the absence of the acceptor, and J is the spectral overlap integral calculated as 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. ... The Quantum Yield of a radiation-induced process is the number of times that a defined event (usually a chemical reaction step) occurs per photon absorbed by the system. ...

where f_D is the normalized donor emission spectrum, and ε_A 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. In analytical chemistry, the molar absorptivity or molar extinction coefficient ε of a chemical species at a given wavelength is a measure of how strongly the species absorbs light at that wavelength. ...

The FRET efficieny relate to the quantum yield and the fluorescence lifetime of the donor molecule as follows:

where τ'_D and τ_D are the donor fluorescence lifetimes in the presence and absence of an acceptor, respectively, or as

where F'_D and F_D are the donor fluorescence intensities with and without an acceptor, respectively.

Methods

Example of FRET between CFP and YFP (Wavelength vs. Absorption): a fusion protein containing CFP and YFP excited at 440nm wavelength. The fluorescent emission peak of CFP overlaps the excitation peak of YFP. Because the two proteins are adjacent to each other, the energy transfer is significant–a large proportion of the energy from CFP is transferred to YFP and creates a much larger YFP emission peak.

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 between CFP and YFP (Wavelength vs. ... Kenyans examining insect-resistant transgenic Bt corn. ... This article or section is not written in the formal tone expected of an encyclopedia article. ... Confocal laser scanning microscopy (CLSM or LSCM) is a valuable tool for obtaining high resolution images and 3-D reconstructions. ... Molecular biology is the study of biology at a molecular level. ... Biophysics (also biological physics) is an interdisciplinary science that applies the theories and methods of physics, to questions of biology. ... Biochemistry is the study of the chemical processes in living organisms. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... A fluorophore is a component of a molecule which causes a molecule to be fluorescent. ... Intermolecular describes a process or characteristic that extends from one molecule to an adjacent one; a property or phenomenon that extends from one molecule to another. ... 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 a...

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. It has been suggested that mGFP be merged into this article or section. ... Kenyans examining insect-resistant transgenic Bt corn. ...

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. Photobleaching is the photochemical destruction of a fluorophore. ... 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. ... Luciferase is a generic name for enzymes commonly used in nature for bioluminescence. ...

FRET and BRET are also the common tools in the study of biochemical reaction kinetics and molecular motors. Kinetics refers to two different areas of science: Chemical kinetics studies reaction rates. ... Molecular motors are biological nanomachines and are the essential agents of movement in living organisms. ...

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. BiFC is a means of observing protein protein interactions. ...

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. Phosgene is a highly toxic chemical compound with the formula COCl2. ... The chemical structure of triphosgene Space-filling model of triphosgene Triphosgene (Bis(trichloromethyl) carbonate, C3Cl6O3) is a chemical compound that is used as a substitute for phosgene. ... Coumarin is a chemical compound/poison found in many plants, notably in high concentration in the tonka bean, woodruff, and bison grass. ... 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. ... For other uses, see Concentration (disambiguation). ...

Image File history File links Size of this preview: 800 × 592 pixelsFull resolution (1003 × 742 pixel, file size: 14 KB, MIME type: image/png) FRET_application_phosgene_detection I, the creator of this work, hereby grant the permission to copy, distribute and/or modify this document under the terms of the GNU Free...

References