Quenching Index Anisotropy

Fluorescent Resonance Energy Transfer (FRET) as a Probe of Proximity in Proteins

FRET is a distance-dependent excited state interaction in which emission of one fluorohore is coupled to the excitation of another. It occurs primarily because the acceptor dipole interacts or resonates with the donor dipole. The use of FRET is to obtain structural maps of complex biological structures, primarily proteins and other macromolecular assemblies such as ribosomes and nucleosomes. Measurements of energy transfer can provide intra- or intermolecular distance data for proteins and their ligands in the range 10-100 Angstrom. Also, FRET can detect change in distance (1-2 Angstrom) between loci in proteins, hence it is a sensitive measure of conformational change.

The distance between two residues in a protein can be determined from measurements of quantum yield or lifetime, if one residue has a fluorescence energy donor, and the other an energy acceptor. The efficiency of energy transfer measures the degree of overlap between the donor emission spectrum and acceptor absorption spectrum. This allows for determination of proximity and relative orientation of the fluorophores. The distance between the two is inversely proportional to the sixth power.

Mechanism of FRET

The excitation energy can be transferred by a radiationless process to a neighboring fluorophore if their energy level difference corresponds to the quantum of excitation energy. In this process, the quantum, or exciton, is transferred, which raises the electron in the acceptor to a higher energy state as the photo-excited electron in the donor returns to ground state. This mechanism requires resonance interaction between donor and acceptor over distances greater than interatomic. The conditions for this mechanism are that the fluorescent emission spectrum of the energy donor overlap the absorption spectrum of the energy acceptor. Also, donor and acceptor transition dipole orientations must be approximately parallel. The probability that energy transfer will occur depends on the sixth power of the distance between the fluorophores. This permits proximity to be measured up to a range of about 10-100 Angstrom. The energy received by the acceptor is less than that given by the donor. The rest of the energy is degraded and is pread over the environment. The acceptor can be fluorescent or non-fluorescent. If the acceptor also is fluorescent, the transferred energy can be emitted as a fluorescence characterisic of the acceptor . If the acceptor is not fluorescent, the energy is lost through equilibration with solvent.

When the donor and acceptor are different, FRET can be detected by the appearence of fluorescence of the acceptor or by quenching of donor fluorescence. When the donor and acceptor are the same, FRET can be detected by the resulting fluorescent depolarization. This energy transfer can be detected and used by measuring an emission of the acceptor fluorophore if it is excited at the donor fluorophore's wavelength. This wavelength normally would not produce an emission from the acceptor, but does if energy transfer is involved. This energy transfer can also be detected by measuring a decrease of donor emission at it's wavelength in the presence of an acceptor. The acceptor has a quenching effect on the donor.

The two fluorophores need not necessarily be part of the same molecule. Energy transfer will take place between isolated molecules in solution as long as the concentration is high enough to bring avarage intermolecular distance within 50-60 Angstrom. The energy transfer is first order in the concentration of the donor, and first-order in the concentration of the acceptor. However, the overall energy transfer is diffusion-controlled in this case. The slow-step is not the second-order transfer itself, but the diffusion.

Foerster Theory

A quantitative theory for singlet-singlet energy transfer has been developed by Theodor Foerster which assumes that the transfer occurs through dipole-dipole interactions of donor and acceptor. This theory is not valid when the donor and acceptor are very close. Multipole and electron exchange interactions can also result in energy transfer.

To obtain useful structure information from energy transfer, the measured efficiency must be related to the distance between the two fluorophores. FRET efficiency, E can be obtained by measuring the fluorescence intensities of the donor with acceptor, Fda and without acceptor, Fd:

E = 1 - Fda/ Fd

FRET efficiency can also be measured using the lifetime of the donor in presence, Tda and absence of the acceptor probe, Td:

E = 1 - Tda / Td 

The relationship between the transfer efficiency and the distance between the donor and acceptor, R is given by the equation:

E = Ro6 / (Ro6 + R6)


R = Ro (1/E - 1)1/6

where Ro is the Foerster distance, that is, the distance at which energy transfer is 50% efficient. In other words, it is the distance where 50% of excited donors are deactivated by FRET. At Ro, there is an equal probability for resonance energy transfer and the radiative emission of a photon. The magnitude of Ro is dependent of the spectral properties of the donor and acceptor:

Ro = [8.8x1023K2n-4QdJ]1/6 (Angstrom)


The donor and acceptor must be within 0.5xRo -1.5xRo from each other. These measurements give the average distance between the two fluorophores. When measuring a change in distance, the result is a scalar and gives no indications of which fluorophore (donor and/or acceptor) moves.

The validity of the Foerster theory was demonstrated with a series of polyproline oligomers having a naphtyl donor at one end and an a dansyl acceptor at the other end:

Dansyl - (Pro)n - NH - CO - NH - Naphtyl

The proline residues formed a polyproline type-II helix (phi = -83, psi = +149) which was confirmed by circular dichroism spectroscopy. Because the distance between naphtyl donor and dansyl acceptor was known from the known dimensions of the rigid helix, measured efficiencies could be compared directly with computed values from the Foerster theory. To measure proximity between various loci of a flexible polypeptide chain, peptides of N-(hydroxyethyl) glutamine were used with the same donor-acceptor pair attached to the ends.

The major uncertainty in the calculation of distance is that kappa square cannot be directly measured. Upper and lower limits can be determined from polarization measurements though. In practical applications, everyone assumes kappa square is 2/3 which is the value obtained for randomly oriented donors and acceptors when both can undergo unrestricted isotropic motion. Experiments can be done with different donor-acceptor pairs. If the same distance is obtained, the assumption is very likely close to correct. Generally, FRET-derived distances aggree within +/- 5 Angstrom with distances obtained from X-ray crystallography.

Donor and Acceptor Pairs

The main difficulty is placing the energy donor and acceptor specifically at known sites in a protein molecule. The finite size of the fluorophores and the occurence of multiple donors and acceptors on the same protein create further difficulties in the establishment of structural maps. Quantitative analysis is restricted to the cases where only a few donors and acceptors are present (ideally one fluoroiphore per protein), or at the other extreme where a large number of acceptors and donors are present so that uniform distributions can be assumed. One such case where uniform distribution maybe achived is where one molecule is uniformly labelled with donors and the another molecule is uniformly labelled with acceptors such as may occur for two polypeptide chains within an oligomeric protein. Generally, however, multiple labeling do not provide the distance between residues but it can still give informations about conformational changes occuring in the molecule.

Some typical donor-acceptor pairs commonly used in structural mapping of proteins, and values of Ro are listed in the table:



Ro (Angstrom)
















Intrinsic fluorophores such as tryptophan or tyrosine have limitations in FRET:

The smallest FRET probes are are transition metal and lanthanide ions which are about 1 Angstrom in diameter. These ions are good acceptors and avoid the problem of uncertasinty in the value of kappa squere.

Energy Transfer Distance Measurements on Actin

Energy transfer has been used to measure distances and to map spatial distribution and assembly within immunoglobulins, rhodopsin, myosin, oligopeptides and between the protein subunits of assemblies ranging from simple oligomeric proteins to ribosomes. Because muscle proteins have well characterized chemistries, FRET has been frequently used to assess intra- and intermolecular distances in their structures. For example, in the Troponin-C / Troponin-I complex (regulators of muscle contraction), distances between six pairs of sites have been determined using FRET.

Here is an example to demonstrate an application of FRET to measure the proximity between tryptophan residues in actin (1atn) and an acceptor probe located at Cys374.

Actin (left) is the main protein component of thin filaments and plays a major role in muscle contraction. Fibrous actin (F-actin) is a helical polimer of a globular protein (G-actin). The atomic structure of actin has been first solved in complex with the enzyme DNase which prevents polymetization . The structure of several other G-actin molecules have been determined later in complex with other inhibitors. The figure shows the structure of G-actin without an inhibitor. The structure has four domains. Two of the domains are similar alpha/beta domains that contain an ATPase catalytic site between them. This ATP is hydrolyzed when G-actin polymerizes to F-actin. Actin belongs to a diverse group of ATP-requiring enzymes that includes hexokinase and chaperone hsc70. These proteins form an evolutionary family that comprises similar three-dimensional structures, the biological functions and amino acid sequences of which have completely diverged.

For determination of transfer efficiency between loci in the protein, actin has been labeled with a non-fluorescent acceptor probe attached to the C-terminal Cys374 cystein. These labels are:

Tryptophan residues are found at positions 79, 86, 340 and 356. The transfer efficiency depends on the sum of all donor-acceptor distances.

Here is a coordinate file for 1atn from the PDBsum database. Follow the appropriate PDB link.

You may want to examine the structure in Rasmol. Note that the entry is a complex of actin and DNase. Three residues from the C-terminus (Lys373, Cys374, Phe375) have been removed by mild tryptic digestion of the complex prior to crystallization. The last residue in the actin coordinate dataset is Arg372. The residue Cys374 that has been labeled with the DDPM and DABMI non-fluorescent acceptors is not in the coordinate file. It is within a few Angstrom to Arg372, though.

Using the Rasmol command line, type the following commands and examine the position of tryptophan residues in relation to each other and to Arg372:

restrict *a
select trp79
color white
select trp86
color yellow
select trp340
color green
select trp356
color blue
select arg372
color red

The measured FRET distances (in Angstrom) between the four tryptophans to Cys374 are as follows:
















Thus FRET appears to very usefully supplement the crystallographic dataset.

FRET Distances in IgG

The overall structure of antibodies has been well established by a variety of different methods. The Y-shaped structure of the IgG molecule contains two arms, Fab, each of which contains an antigen binding site. It also contains a third part, Fc, which is important in other aspects of antibody function. This structure is made up of four different polypeptides, two heavy chains and two light chains which are linked through disulfide bonds. The Fc portion of the molecule can be removed by enzymatic digestion with pepsin to give a stable molecule containing the two linked Fab' arms, F(ab')2.

The structure of IgG has been studied using FRET. Three different distances were measured:

The measured distances are in good agreement (within +/- 5 Angstrom) with the results of several other studies, including x-ray crystallography of various immunoglobuline molecules.

Quenching Index Anisotropy