Proteins, fluorescence anisotropy

Shi X, Basran J, Seward FIE, Childs W, Bagshaw CR, Boxer SG (2007) Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C) quantitative analysis of FRET in YFP dimers. Biochemistry 46 14403-14417... 

J. R. Lakowicz, G. Laszko, I. Gryczynski, and H. Cherek, Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry, J. Biol. Chem. 261, 2240-2245 (1986). 

J. R. Lakowicz and G. Weber, Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies, Biophys. J. 32, 591-601 (1980). 

The fluorescence lifetime is sensitive to the environment of the fluorophore, and in membranes this usually means the surrounding fatty acyl chains or the membrane protein interfacial region (see summary in Table 5.3). Generally, the lifetime of membrane-bound fluorophores is rather less sensitive to the types of subtle alterations which are encountered in membranes as compared to the fluorescence anisotropy parameters. The gel-to-liquid crystalline phase transition is a notable exception where most fluorophores show an alteration in lifetime properties. Although, again, the anisotropy (see below) is the most sensitive parameter in this regard, the fluorescence lifetime has been used with considerable success in the study of phase transitions and lateral phase separations. Fluorophores used to yield information on the... 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

F. Jahnig, Structural order of lipids and proteins in membranes Evaluation of fluorescence anisotropy data, Proc. Natl. Acad. Sci. U.SA. 76, 6361-6365 (1979). 

H. Fukumura and K. Hayashi, Time-resolved fluorescence anisotropy of labeled plasma proteins adsorbed to polymer surfaces, J. Colloid Interface Sci. 135, 435M42 (1990). 

Fig. 10 Binding curves obtained by measuring the electrophoretic mobility ( ) and fluorescence anisotropy (o) of F-ll-mer at varying concentrations of SSB protein in the running buffer. Conditions were the same as for Figure 9. (From Ref. 48.)...

Steady-state measurements of the fluorescence anisotropy of fluorescein derivatives form the basis of a sensitive analytical technique also used to detect and quantitate proteins [36], steroids [37-39], therapeutic drugs, and narcotics [40-42], In a different approach, the anisotropy of the fluorescein conjugate is measured as a function of the medium viscosity to determine the segmental mobility of the chains that cover the binding site [43-45],... 

Also, high-throughput screening of small ligands for proteins [53] or other biomacromolecules of therapeutic interest can be done with the aid of aptamers. Because small molecules replace an ap tamer in its complex with the macromolecule, this substitution can be monitored by a change of fluorescence anisotropy of labeled ap tamer or by a change in the activity of an aptazyme. 

Brownbridge, G. G., Lowe, P. N., Moore, K. J., Skinner, R. H., and Webb, M. R. (1993). Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method. Essential role of ARG-903 of GAP in activation of GTP hydrolysis on p21ras. J. Biol. Chem. 268, 10914-10919. 

LiCata, V.J., and Wowor, A. J. (2008). Applications of fluorescence anisotropy to the study of protein-DNA interactions. Methods Cell Biol. 84, 243—262. 

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