Quenching emission anisotropy

In general, when anisotropy varies as a function of quencher concentration, the Perrin plot can be written as 

Measiuements of the emission anisotropy A as a function of added collisional quencher are made with the steady fluorescence intensity, which integrates the different weighted fluorescence lifetimes. Quenching emission anisotropy plot of 1 /A vs I (Fig. 5.14) yields for A(o) a value of 0.246 and 0.243 for [L-Met2] DREK and DREK, respectively. These values, lower than that (0.278) measured at - 45 °C for tyrosine at 280 nm (Lakowicz and Maliwal, 1983), indicate that tyrosine residue in both peptides display residual motion independent of the global rotation of the peptide. It is possible to measure the relative importance of the mean residual motions of the tyrosine residues  

The fluorescence anisotropy A of a fluorescent probe in a molecule can be given by 

The value of Op found is equal to 1.1 ns, a value in good agreement with the 1 ns value suggested for these peptides from NMR experiments (Pastore et al. 1985). 

Fluorescence anisotropy decay of [Leu ] enkephalin tyrosine was measured using the frequency- domain up to 10 GHz. The data indicate a 44 ps cori elation time for local tyrosine motions and a 219 ps correlation time for overall rotational diffusion of the pentapeptide (Lakowicz et al. 1993). Also a rotational correlation time of 26 ps was measured by H NMR for Ha of tyrosine in position 1 of L-dermorphin (Simenel, 1990). These ps values determined by NMR and by fluorescence spectroscopy are the result of possible significant atomic fluctuations that occur in the picosecond time scale (Karplus and Me Gammon, 1981). Since it was difficult in quenching experiments performed on DREK to measure such short correlation times we do not know whether these atomic fluctuations would depend on the conformation of the peptide or not. However, our results clearly put into evidence the presence of a local rotation within DREK. 

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Table 9.6. Rotational correlation time ( )p of Mb obtained according to the classical Perrin plot, to the quenching emission anisotropy and to the theoretical equation.

Homotransfer does not cause additional de-excitation of the donor molecules, i.e. does not result in fluorescence quenching. In fact, the probability of de-excitation of a donor molecule does not depend on the fact that this molecule was initially excited by absorption of a photon or by transfer of excitation from another donor molecule. Therefore, the fluorescence decay of a population of donor molecules is not perturbed by possible excitation transport among donors. Because the transition dipole moments of the molecules are not parallel (except in very rare cases), the polarization of the emitted fluorescence is affected by homotransfer and information on the kinetics of excitation transport is provided by the decay of emission anisotropy. 

There should exist a correlation between the two time-resolved functions the decay of the fluorescence intensity and the decay of the emission anisotropy. If the fluorophore undergoes intramolecular rotation with some potential energy and the quenching of its emission has an angular dependence, then the intensity decay function is predicted to be strongly dependent on the rotational diffusion coefficient of the fluorophore.(112) It is expected to be single-exponential only in the case when the internal rotation is fast as compared with an averaged decay rate. As the internal rotation becomes slower, the intensity decay function should exhibit nonexponential behavior. 

M. Eftink, Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins, Biophys. J. 43, 323-334 (1983). 

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 mean fluorescence lifetime is used to calculate the rotational correlation time from the Perrin plot (quenching resolved emission anisotropy experiment). 

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. 

Quenching resolved emission anisotropy experiments could be performed at emission wavelengths in the blue (< 330 nm) and red (> 330 nm) portions of the spectrum to yield a more consistent data surface. However, this could be possible if at each edge of the fluorescence spectrum the emission occurs mainly from the buried or the surface Trp residues. Unfortunately, this is not the case since for example at 315 nm, the fractional contribution to the total fluorescence of the surface Trp residue is 42%. 

Fluorescence quenching experiments with oxygen produce emission with non-exponential dec s. So, measuremeDts of the emission anisotropy as a function of added coUisional quencher were made with the steady fluorescence intensity, which integrates the different weighted fluorescence lifetimes, lypical quenchirtg emission anisotropy plots of 1 / A versus F/Fo of Mb " are shown in Fig. 9.15. The plots are linear to within experimental error and can be described hy the simplified relation ... 

Albani, J. R, 1999, New insights in the conformation of a i-acid glycoprotein (orosomucoid). Quenching resolved emission anisotropy studies. Spectrochimica Acta, Part A. 55,2353-2360. 

Several molecular properties can be measured using emission and excitation spectra. These include fluorescence lifetime, efficiency, anisotropy of the emitted light, mobility of chromophores, rates of quenching, and energy transfer to other chromophores. 

Fig. 10. Highly schematic representation of the orientation of several tryptophan-containing peptides with respect to calmodulin. (A) With tryptophan in position 1, the indole is located on the hydrophilic side of the helix and is exposed to solvent. Peptides with tryptophan on this face of the helix should exhibit emission maxima near that of indole in water ( 350 nm), a small anisotropy, and a high accessibility for acrylamide quenching. (B) In position 2, the tryptophan is partially exposed at the interface between the peptide and calmodulin. Peptides with a tryptophan in this location should have fluorescence properties that are intermediate between example A and C. (C) The tryptophan is on the hydrophobic side of the helix and is almost entirely buried. The emission maximum should be strongly blue-shifted, the anisotropy should be large, and the accessibility to acrylamide quenching low. Taken from O Neil et al. (1987).

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