Anisotropy quenching method

The structural dimension at a water/DCE interface is d — 2.48, while short-range structural information about the interface obtained by the fluorescence dynamic anisotropy experiments suggests that the interface is three-dimensional-like. Taking the results obtained by molecular dynamics simulations into account, these results can be understood only by the fact that the water/DCE interface is thin ( 1 nm), but is rough with respect to the spatial resolution of the excitation energy transfer quenching method ( 7 nm), as shown in Figure 12.7. 

The long lifetime of phosphorescence allows it to be used for processes which are slow—on the millisecond to microsecond time scale. Among these processes are the turnover time of enzymes and diffusion of large aggregates or smaller proteins in a restricted environment, such as, for example, proteins in membranes. Phosphorescence anisotropy is one method to study these processes, giving information on rotational diffusion. Quenching by external molecules is another potentially powerful method in this case it can lead to information on tryptophan location and the structural dynamics of the protein. 

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. 

Additionally, since the acceptor is excited as a result of FRET, those acceptors that are fluorescent will emit photons (proportional to their quantum efficiency) also when FRET occurs. This is called sensitized emission and can also be a good measure of FRET (see Fig. 1). To quantitate FRET efficiency in practice, several approaches have been evolved so far. In flow cytometric FRET (7), we can obtain cell-averaged statistics for large cell populations, while the subcellular details can be investigated with various microscopic approaches. Jares-Erijman and Jovin have classified 22 different approaches that can be used to quantify energy transfer (8). Most of them are based on donor quenching and/or acceptor sensitization, and a few on measuring emission anisotropy of either the donor or the acceptor. Some of these methods can be combined to extend the information content of the measurement, for example two-sided FRET (9) involves both acceptor depletion (10) and... 

The theory of high order corrections to the Lamb shift described above for H and D may also be applied to other light hydrogenlike ions. The simplest such ion is He+. Originally the classic Lamb shift in 17e+ was measured in [50] by the quenching-anisotropy method with the result L 2Si — 2Pi,17e+) = 14 042.52 (16) MHz. Later the authors of [50] discovered a previously unsuspected source of systematic error in their experiment. Their new measurement of the classic Lamb shift in 17e+ by the anisotropy method resulted in the value L 2Si — 2Pi,He ) = 14 041.13 (17) MHz [51]. Besides the experimental data this result depends also on the theoretical value of the hne structure interval. In [51] the value AE 2Pz —2Pi) = 175 593.50 (2) MHz was used. We recalculated this interval using tire latest theoretical results discussed above and obtained AE 2P3 — 2Pi) = 175 593.33 (1) MHz. Then the value of the... 

When a protein contains fluorophore residues located at the surface and in the core as it is the case for a j-acid glycoprotein, the results obtained from the classical Perrin plot contain contributions from all residues. In order to obtain information on the motion of each class of fluorophore residues, one may follow anisotropy and intensity variations as a function of quencher concentration (Quenching Resolved Emission Anisotropy) or / and anisotropy and lifetime variations with temperature (-50 to +35°C) (Weber method). 

Table 8.7. Comparison of the anisotropies of the two classes of Trp residues of ai-acid glycoprotein and Lens culinaris agglutinin. Measurements were performed at 20°C with the Quenching Resolved Emission Anisotropy method.

Although we haye shown that the three Trp-residues contribute to the global fluorescence of a i-acid glycoprotein (paragraph 5 c of this chapter) quenching resolved emission anisotropy and the Weber method allowed giving a description on the mean local dynamic of the Trp-residues. The dynamic of the surface Trp residue is well separated from the two other Trp residues. 

Some of these applications are complementary to the steady state methods discussed in section 8.2. Investigations of fluorescence lifetimes and of anisotropy or fluorescence quenching phenomena in the lifetime mode, that is during the decay after a single flash, require more elaborate instrumentation and theory than steady state investigations. On the whole applications rather than detail of methods are discussed here. The use of the lifetime method for the study of molecular rotation, domain movement and more local dynamic events can often, some experts say always, provide additional information even for those problems which can be investigated with considerable success by steady state measurements. 

Comparison between scaled theoretical and experimental one-electron Lamb shifts, expressed as deviations from the theoretical mjjn. The upper horizontal line for each ion is Erij son s theory and the lower horizontal line is Mohr s theory and S is the average of the two. The experimental data are labelled by the method of measurement according to (0) microwave resonance (a) anisotropy measurement (+) quench-rate measurement ( ) laser resonance. The dashed lines are the Borie corrections to the theoretical values. (From Drake )... 

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