Spectroscopic ruler

These and other workers(46) have proposed that the energy transfer process can serve as a spectroscopic ruler (10-60 A) in biological systems. 

Stryer, L. (1978). Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819—46. 

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 use of Forster non-radiative energy transfer for measuring distances at a supramolecular level (spectroscopic ruler) will be discussed in detail in Chapter 9. 

The Forster resonance energy transfer can be used as a spectroscopic ruler in the range of 10-100 A. The distance between the donor and acceptor molecules should be constant during the donor lifetime, and greater than about 10 A in order to avoid the effect of short-range interactions. The validity of such a spectroscopic ruler has been confirmed by studies on model systems in which the donor and acceptor are separated by well-defined rigid spacers. Several precautions must be taken to ensure correct use of the spectroscopic ruler, which is based on the use of Eqs (9.1) to (9.3) ... 

Chapter 9 is devoted to resonance energy transfer and its applications in the cases of donor-acceptor pairs, assemblies of donor and acceptor, and assemblies of like fluorophores. In particular, the use of resonance energy transfer as a spectroscopic ruler , i.e. for the estimation of distances and distance distributions, is presented. 

Energy transfer has been used extensively in biological work as a spectroscopic ruler (45 1 and in numerous other studies1-46" the underlying assumption being that the Forster expressions are valid in all situations. 

Stryer L, Haugland RP (1967) Energy transfer a spectroscopic ruler. Proc Nat Acad Sci USA 58 719-726... 

L. Stryer, Fluorescence Energy Transfer as a Spectroscopic Ruler, Annu. Rev. Biochem. 1978,47, 819 C. Berney and G. Danuser, FRET or No FRET A Quantitative Comparison, Biophys. J. 2003, 84, 3992 http //www.probes.com/ handbook/. 

Forster s theory [1], has enabled the efficiency of EET to be predicted and analyzed. The significance of Forster s formulation is evinced by the numerous and diverse areas of study that have been impacted by his paper. This predictive theory was turned on its head by Stryer and Haugland [17], who showed that distances in the range of 2-50 nm between molecular tags in a protein could be measured by a spectroscopic ruler known as fluorescence resonance energy transfer (FRET). Similar kinds of experiments have been employed to analyze the structure and dynamics of interfaces in blends of polymers. 

L. Stryer and R. Haugland, Energy transfer A spectroscopic ruler, Proc. Natl. Acad. Sci. USA 58, 719 (1967). 

The constant Rq is dependent on several parameters 1) the relative orientation of the transition dipole moments of the two molecules (these dipoles are the spectroscopic transition dipoles), 2) the extent that the fluorescence spectrum of the donor overlaps with the absorption spectrum of the acceptor, and 3) the surrounding index of refraction. We will deal with each of these below (see Equation 8). Because many proteins have diameters less than lOnm, this distance dependence explains the usefulness of ERET for determiiung distances inside proteins as well as between interacting proteins, which is the reason that the name spectroscopic ruler was coined for FRET (20). ERET is a convenient method for determining the distance between two locations on proteins, or for determining whether two proteins interact intimately with each other. Fluorescence instrumentation is available in many laboratories, and a plethora of dyes and a wide variety of fluorescent proteins are now readily available. Therefore, FRET is a viable option for most researchers. With care, FRET can yield valuable information concerning protein-protein interactions, interactions of proteins with other molecules, and protein conformational changes. 

M. Bates, T.R. Blosser, X. Zhuang, Short-range spectroscopic ruler based on a single-molecule optical switch. Phys. Rev. Lett. 94, 108101 (2005)... 

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]. 

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. 

Table III summarizes rate, helicity, and donor-acceptor distance data for the 16-mer bundle. Because of the observable trend in conformation versus rate in the electron transfer studies, it was decided to measure donor-acceptor distances in the 16-mer metalloprotein bundles. In order to study the effects of solution conditions on H, the donor-acceptor distance, Forster energy transfer was used as a spectroscopic ruler, according to...

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