Resonance energy transfer distance measurement

Heyduk, E. and Heyduk, T. Thiol-reactive, luminescent europium chelates Luminescence probes for resonance energy transfer distance measurements in biomolecules. Anal. Biochem. 248 216-227, 1997. 

The intramolecular distances measured at room temperature with the AEDANS FITC pair were similar in the Ca2Ei and E2V states [297]. Ca and lanthanides are expected to stabilize the Ej conformation of the Ca -ATPase, since they induce a similar crystal form of Ca -ATPase [119,157] and have similar effects on the tryptophan fluorescence [151] and on the trypsin sensitivity of Ca -ATPase [119,120]. It is also likely that the vanadate-stabilized E2V state is similar to the p2 P state stabilized by Pi [418]. Therefore the absence of significant difference in the resonance energy transfer distances between the two states implies that the structural differences between the two conformations at sites recorded by currently available probes, fall within the considerable error of resonance energy transfer measurements. Even if these distances would vary by as much as 5 A the difference between the two conformations could not be established reliably. 

This chapter reviews several techniques which combine the use of laser microbeams with antibodies to study molecular and cellular biology. An overview of the basic properties of lasers and their integration with microscopes and computers is provided. Biophysical applications, such as fluorescence recovery after photobleaching to measure molecular mobility and fluorescence resonance energy transfer to measure molecular distances, as well as ablative applications for the selective inactivation of proteins or the selective killing of cells are described. Other techniques, such as optical trapping, that do not rely on the interaction of the laser with the targeting antibody, are also discussed. 

Sabanayi am, CR, Fid, JS, and Meller, A, Using fluorescence resonance energy transfer to measure distances along individual DNA molecules Corrections due to nonideal transfer. Journal of Chemical Physics 122 (2005) art. no.-061103. 

Fluorescence resonance energy transfer (FRET) is a technique that has been used to measure distances between pairs of proximal fluorochromes. A suitable pair consists of a donor fluorochrome, which has an emission spectrum... 

Fluorescence resonance energy transfer (FRET) is a spectroscopic means of obtaining distance information over a range up to 80 A in solution. It is based on the dipolar coupling between the electronic transition moments of a donor and acceptor fluorophore attached at known positions on the RNA species of interest. It can be applied in ensembles of molecules, either by steady-state fluorescence or by lifetime measurements, but it is also very appropriate for single-molecule studies. In addition to the provision of distance information, recent studies have emphasized the orientation dependence of energy transfer. 

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. 

---