Proteins fluorescence energy transfer

Young, R., Arnette, J., Roess, D. and Barisas, B. (1994). Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics. Biophys. J. 67, 881-8. 

Verbist, J., Gadella, T. W. J., Raeymaekers, L., Wuytack, F., Wirtz, K. W. A. and Casteels, R. (1991). Phosphoinositide-protein Interactions of the plasma-membrane Ca2+-transport ATPase as revealed by fluorescence energy-transfer. Biochim. Biophys. Acta 1063, 1-6. 

The fluorescence energy transfer process has been widely used to determine the distance between fluorophores, the surface density of fluorophores in the lipid bilayer, and the orientation of membrane protein or protein segments, often with reference to the membrane surface and protein-protein interactions. Membranes are intrinsically dynamic in nature, so that so far the major applications have been the determination of fixed distances between molecules of interest in the membrane. 

Fluorescence energy transfer. Panel (a) shows the fluorescence characteristics of a hypothetical protein A (excitation and emission maxima at 290 nm and 350 nm, respectively). In (b). Ligand B fluoresces with an emission maximum at 450 nm when excited at 350 nm. Panel (c) shows that the formation of the protein-ligand complex can be monitored using an excitation wavelength of 290 nm and recording the decrease in protein fluorescence at 350 nm or the increase in ligand fluorescence at 450 nm... 

Horton, R.A. et al. 2007. A substrate for deubiquitinating enzymes based on time-resolved fluorescence energy transfer between terbium and yellow fluorescent protein. Anal. Biochem. 360, 138-143. 

From the mere fact that CF, can be released from the membrane by EDTA treatment and the enzyme stays in solution without detergents, it is apparent that the catalytic sector has minimal, if any, direct interaction with the lipids of the chloroplast membrane. It is a globular protein that is held to the surface of the membrane via interaction with the membrane sector. Recently it was shown that the y subunit is in immediate contact with the membrane sector and the 8 and e subunits may induce proper binding for catalysis [17,18], The enzyme contains a few well-defined sites that were used for localization experiments by the method of fluorescent energy transfer [19,56-61], These studies revealed the position of those sites and helped to localize the various subunits of CF, in space relative to the chloroplast membranes (for a model of CF, see Refs. 61 and 62). These experiments are awaiting analysis of the amino acid sequence of the y subunit that is now under investigation in Herrmann s laboratory [148], Definite structural analysis could be obtained only after good crystals of the enzyme become available. 

Fig. 14. Isotope effect on the rate of hydride transfer. The rate of hydride transfer to dihydrofolate catalyzed by dihydrofolate reductase (IS /rAf) was measured by fluorescence energy transfer, exciting the protein at 280 nm and observing emission by NADPH at 450 nm. The reaetion with NADPH oecurred at a rate of 450 see", followed by a linear phase at 12 sec , as shown by the smooth line. The rate of the burst observed with NADPD, the deuterium analog, occurred at 150 sec . Reproduced with permission from (27).

The application of total internal reflection fluorescence spectroscopy (TIRF) by this laboratory to the study of protein adsorption at solid-liquid interfaces is reviewed. TIRF has been used to determine adsorption isotherms and adsorption rates from single-and multi-component protein solutions. Initial adsorption rates of BSA can be explained qualitatively by the properties of the adsorbing surface. Most recently, a TIRF study using monoclonal antibodies to probe the conformation of adsorbed sperm whale myoglobin (Mb) elucidated two aspects of the Mb adsorption process 1) Mb adsorbs in a non-random manner. 2) Conformational changes of adsorbed Mb, if they occur, are minor and confined to local regions of the molecule. Fluorescence energy transfer and proteolytic enzyme techniques, when coupled with TIRF, can characterize, respectively, the conformation and orientation of adsorbed Mb. 

The recent investigation of Mb adsorption on PDMS (above) has probed the conformation and orientation of a layer of protein adsorbed on a hydrophobic surface. Our present goal is further characterization of the conformation and orientation of Mb on PDMS. Two techniques, fluorescence energy transfer and proteolytic enzyme cleavage, can be used to achieve this goal. 

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