Fluorescent proteins localize the guanosine 5'-triphosphate hydrolase ARF in the Golgi apparatus of a living macrophage. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl Guanosine is a Nucleoside comprising Guanine attached to a Ribose ( Ribofuranose) ring via a β-N9- Glycosidic bond. Guanosine-5'-triphosphate ( GTP) is a Purine Nucleotide. One role is as substrate for the synthesis of RNA during transcription. In Biochemistry, a hydrolase is an Enzyme that catalyzes the Hydrolysis of a Chemical bond. The Golgi apparatus (also called the Macrophages ( Greek: "big eaters" from makros "large" + phagein "eat" ( Mø) are cells within the tissues that FRET studies revealed ARF activation in the Golgi and in the formation of phagosomes. [1]

Förster resonance energy transfer (abbreviated FRET) describes an energy transfer mechanism between two chromophores. 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). In physics there are two kinds of dipoles ( Hellènic: di(s- = two- and pòla = pivot hinge An electric dipole is a This energy transfer mechanism is termed Förster resonance energy transfer , named after the German scientist de:Theodor Förster[2]. When both molecules are fluorescent, the term "fluorescence resonance energy transfer" is often used, although the energy is not actually transferred by fluorescence. Fluorescence is a Luminescence that is mostly found as an [3],[4] 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," although the latter enjoys common usage in scientific literature.

## Theoretical basis

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

$E = \frac{k_{ET}}{k_f+k_{ET}+\sum{k_i}}$

where kET is the rate of energy transfer, kf the radiative decay rate and the ki 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. 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 A material's absorption spectrum shows the fraction of incident Electromagnetic radiation absorbed by the material over a range of Frequencies.
• 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:

$E=\frac{1}{1+(r/R_0)^6}\!$

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:

${R_0}^6 = 8.8 \times 10^{-28} \; \kappa^2 \, n^{-4} \, Q_0 \, J$

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

$J = \int f_{\rm D}(\lambda) \, \epsilon_{\rm A}(\lambda) \, \lambda^4 \, d\lambda$

where fD is the normalized donor emission spectrum, and εA is the acceptor molar extinction coefficient. The European Space Agency 's INTErnational Gamma-Ray Astrophysics Laboratory ( INTEGRAL) is detecting some of the most energetic radiation that comes from space 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 occurs per Photon absorbed by the system The molar extinction coefficient, also known as molar absorptivity, is a measurement of how strongly a Chemical species absorbs light at a given 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:

$E = 1 - {\tau'_{\rm D}}/{\tau_{\rm D}} \!$

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

$E = 1 - {F'_{\rm D}}/{F_{\rm D}} \!$

where F'D and FD 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. Genetic engineering, Recombinant DNA technology, genetic modification/manipulation (GM and gene splicing are terms that apply to the direct 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. Microscopy is the technical field of using microscopes to view samples or objects Confocal laser scanning microscopy ( CLSM or LSCM) is a technique for obtaining high- resolution optical images Molecular biology is the study of Biology at a molecular level Biophysics (also biological physics) is an Interdisciplinary Science that employs and develops theories and methods of the Physical sciences for Biochemistry is the study of the chemical processes in living Organisms It deals with the Structure and function of cellular components such as Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known 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. A fluorophore, in analogy to a Chromophore, is a component of a molecule which causes a molecule to be Fluorescent. 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. In Physics, Chemistry, and Biology, intermolecular forces are forces that act between stable Molecules or between functional groups of 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. In Chemistry, a ligand is either an Atom, Ion, or Molecule (see also Functional group) that bonds to a central metal generally

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). The green fluorescent protein ( GFP) is composed of 238 Amino acids (26 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. Genetic engineering, Recombinant DNA technology, genetic modification/manipulation (GM and gene splicing are terms that apply to the direct 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. Photobleaching is the photochemical destruction of a Fluorophore. To avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. 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 This technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than CFP to produce an initial photon emission compatible with YFP. 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. Molecular motors are biological Molecular machines that are the essential agents of movement in living organisms

### Other methods

A different, but related, mechanism is Dexter Electron Transfer. Dexter electron transfer is a method for an excited electron state to transfer from one molecule (the donor to a second (the acceptor

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. Bimolecular fluorescence complementation ( BiFC) is a method of viewing the association of Proteins inside living cells. 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. Phosgene is the Chemical compound with the formula COCl2 This colorless gas gained infamy as a Chemical weapon during World War I In it, phosgene or rather triphosgene as a safe substitute serves as a linker between an acceptor and a donor coumarine (forming urea groups). Triphosgene ( Bis(trichloromethyl carbonate, C3Cl6O3 is a chemical compound that is used as a substitute for Phosgene, because at Coumarin is a Chemical compound ( Benzopyrone) a Toxin found in many Plants notably in high concentration in the Tonka bean, Urea is an Organic compound with the Chemical formula ( N[[hydrogen H]]22 C[[oxygen O]] [5] The presence of phosgene is detected at 5x10-5M with a typical FRET emission at 464 nm. In Chemistry, concentration is the measure of how much of a given substance there is mixed with another substance

FRET is also used to study lipid rafts in cell membranes. A lipid raft is a Cholesterol -enriched microdomain in Cell membranes Properties of lipid rafts Rietveld & Simons related lipid rafts in model The cell membrane (also called the plasma membrane, plasmalemma, or "phospholipid bilayer" is a Selectively permeable Lipid bilayer