Spectroscopic Ruler Technique

Once Ra has been determined for a particular D and A pair, it is possible to estimate, experimentally, the actual separation distance (r) in a particular system, via Equation 2.22, prompting the use of the term Spectroscopic Ruler Technique to describe such measurements. 

FRET is an extremely useful phenomenon when it comes to the analysis of molecular conformations and interactions. F or the analysis of interactions, in which two separate molecules are labeled with an appropriate pair of fluorophores, an interaction can be shown by observing FRET. Further, FRET can be used as a type of spectroscopic ruler to measure the closeness of interactions. Proteins, lipids, enzymes, DNA, and RNA can all be labeled and interactions documented. This general method can be applied not only to questions of cellular function like kinase dynamics [3] but also to disease pathways, for example, the APP-PS1 interaction that is important in Alzheimer s disease (AD) [4], Alternatively, two parts of a molecule of interest can be labeled with a donor and acceptor fluorophore. Using this technique, changes in protein conformation and differences between isoforms of proteins can be measured, as well as protein cleavage. 

The distance scale on which FRET occurs makes the technique very attractive in the life sciences because it corresponds well to relevant distances in biology for example, the distance between base pairs in double-stranded DNA is 0.3 nm. The potential of FRET to reveal proximity in biological macro molecules was already pointed out in 1967 by Stryer and Haugland in their article Energy Transfer A Spectroscopic Ruler [98]. In their pioneering experiment, they labeled poly-pro-line peptides of different lengths at both ends and demonstrated the R dependence of the energy-transfer efficiency. Today, FRET is a weU-established spectroscopic technique [57, 58]. For a review, see the article by Selvin [99]. 

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