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Bioluminescence Resonance Energy Transfer (BRET)

Bioluminescence resonance energy transfer(BRET) uses the FRET effect with native

fluorescent or luminescent proteins as chromophores. The phenomenon is observed

naturally for example with the sea pansyRenilla reniformis. It contains the enzyme

Example 3FRET APPLICATIONS IN DNA SEQUENCING AND INVESTIGATION
OF MOLECULAR MECHANISMS

BigDyes™are a widely used application of FRET fluorophores (Fig. 12.12). Since

1997, these fluorophores are generally used as chain termination markers in

automated DNA sequencing. As such, BigDyes™are in major parts responsible

for the great success of genome projects.

In many instances, FRET allows monitoring of conformational changes, protein

folding, as well as protein–protein, protein–membrane and protein–DNA

interactions. For instance, the three subunits of T4 DNA polymerase holoenzyme

arrange around DNA in torus-like geometry. Using the tryptophan residue in one of

the subunits as FRET donor and a coumarine label conjugated to a cysteine residue

in the adjacent subunit (FRET acceptor), the distance change between both subunits

could be monitored and seven steps involved in opening and closing of the

polymerase could be identified. Other examples of this approach include studies

of the architecture ofEscherichia coliRNA polymerase, the calcium-dependent

change of troponin and structural studies of neuropeptide Y dimers.

Me 2 NONMe 2

Cl COO–

Cl

O NH

O O COO–

O

N

H

NH

O

O–

+

O

O

O

N

O H O

N

O

PO 2 –

PO 2 –

O

PO 2 –

HO

Fig. 12.12Structure of one of the four BigDye™terminators, ddT-EO-6CFB-dTMR. The moieties from

left to right are: 5-carboxy-dichloro-rhodamine (FRET acceptor), 4-aminomethyl benzoate linker,

6-carboxy-4^0 -aminomethylfluorescein (FRET donor), propargyl ethoxyamino linker, dUTP.

502 Spectroscopic techniques: I Photometric techniques