Probes resonance energy transfer

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. 

Takakusa H, Kikuchi K, Urano Y, Kojima H, Nagano T (2003) A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral. Chemistry 9 1479-1485... 

Hagiwara Y, Hasegawa T, Shoji A et al (2008) Acridone-tagged DNA as a new probe for DNA detection by fluorescence resonance energy transfer and for mismatch DNA recognition. Bioorg Med Chem 16 7013-7020... 

The lifetime of the excited state of fluorophores may be altered by physical and biochemical properties of its environment. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful analytical tool for the quantitative mapping of fluorescent molecules that reports, for instance, on local ion concentration, pH, and viscosity, the fluorescence lifetime of a donor fluorophore, Forster resonance energy transfer can be also imaged by FLIM. This provides a robust method for mapping protein-protein interactions and for probing the complexity of molecular interaction networks. 

Sinev, M., Landsmann, P., Sineva, E., Ittah, V. and Haas, E. (2000). Design consideration and probes for fluorescence resonance energy transfer studies. Bioconjug. Chem. 11, 352-362. 

Grunewald, J., Kopp, F., Mahlert, C., Linne, U., Sieber, S. A. and Marahiel, M. A. (2005). Fluorescence resonance energy transfer as a probe of peptide cyclization catalyzed by nonribosomal thioesterase domains. Chem. Biol. 12, 873-881. 

Didenko, V. V. (2001). DNA probes using fluorescence resonance energy transfer (FRET) Designs and applications. Biotechniques 31,1106-1116. 

Okamura, Y. and Watanabe, Y. (2006). Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals. Methods Mol. Biol. 335, 43-56. 

Dennis, A. M. and Bao, G. (2008). Quantum dot-fluorescent protein pairs as novel fluorescence resonance energy transfer probes. Nano Lett. 8, 1439-45. 

An increase in sensitivity and reliability of chip analysis can also be achieved by using fluorescence resonance energy transfer (FRET). For this purpose both the probe and the target are labeled with a fluorophor. When the emission spectrum of the donor, e.g. Cy5, overlaps with the absorption spectrum of the acceptor, e.g. Cy5.5, and the donor and the acceptor are at a certain distance from each other, energy is transferred from the donor to the acceptor on excitation of the donor fluorophor. 

The already critical need for molecular-scale compositional mapping will increase as more complex structures are assembled. Currently, electron microscopy, scanning probe microscopy (SPM) and fluorescence resonance energy transfer (FRET) are the only methods that routinely provide nanometer resolution. 

The mechanisms of resonance energy transfer (RET) between a donor and an acceptor have been described in Section 4.6.3. The aim of this chapter is to present further aspects of RET and its applications for probing matter or living systems. RET is particularly widely used to determine distances in biomolecules and supra-molecular associations and assemblies. 

Figure 10.12. Absorption spectra of Phenol Red and emission spectrum of Eosin as potential pH probe based on resonance energy transfer.

Fluorescence resonance energy transfer has also been used for ionic strength measurements.(95) Fluorescein labeled dextran (donor) and polyethyleneimine-Texas Red (acceptor) were placed behind a dialysis membrane. The polymer association is ionic strength dependent and the ratio of intensities (F o/Fw) was used as the measured parameter. Since both the donor and acceptor are fluorescent, this kind of sensor may allow expand the sensitive ionic strength range by shifts in observation wavelength, as was discussed for pH probe Carboxy SNAFL-2 (see Section 10.3). 

P. S. Uster and R. E. Pagano, Resonance energy transfer microscopy Observations of membrane-bound fluorescent probes in model membranes and in living cells, J. Cell. Biol. 103, 1221-1234 (1986). 

Fluorescence resonance energy transfer (FRET) occurs when the excited state energy is transferred from a donor (D) to an acceptor (A). In general, probes containing two separate molecules can be synthesized, a fluorophore (D) and an analyte-sensitive acceptor (A) as shown below ... 

Modification of Cells for Transport Experiments Experimental control of intracellular environment, 171, 817 implantation of isolated carriers and receptors into living cells by Sendai virus envelope-mediated fusion, 171, 829 resonance energy transfer microscopy visual colocalization of fluorescent lipid probes in liposomes, 171, 850. 

Koo, K., and Jaykus, L.-A. (2003). Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an symmetric fluorogenic probe set. Appl. Environ. Microbiol. 69,1082-1088. 

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