Fluorophore rotational freedom

The rotation of the fluorophores is a factor that affects the energy transfer. Only maximal rotational freedom will permit tda estimation. There is no way to predict this factor. Therefore the dynamic averaged value of k2 is considered 2/3. This prediction induces a certain error in the calculation of distances (see Chap. 1). 

Finally, it should be noted that homo-FRET, which is just the exchange of energies between the same dyes, is undetected by common spectroscopic or lifetime measurements and needs the hetero-FRET probing for its detection. The Red-Edge effect allows the easy distinguishing of the decrease of anisotropy due to FRET (static effect) from that occurring due to rotational freedom of fluorophores (dynamic effect), which does not depend on excitation wavelength. 

One can employ linearly polarized light to excite selectively those fluorophores that are in a particular orientation. The difference between excitation and emitted light polarization changes whenever fluorophores rotate during the period of time between excitation and emission. The magnitude of depolarization can be measured, and one can therefore deduce the fluorophore s rotational relaxation kinetics. Extrinsic fluorescence probes are especially useful here, because the proper choice of their fluorescence lifetime will greatly improve the measurement of rotational relaxation rates. One can also determine the freedom of motion of the probe relative to the rotational diffusion properties of the macromolecule to which it is attached. When held rigidly by the macromolecule, the depolarization of a probe s fluorescence is dominated by the the motion of the macromolecule. 

Fluorescent compounds are sensitive to changes in their chemical environment. Alterations in media pH, buffer components, solvent polarity, or dissolved oxygen can affect and quench the quantum yield of a fluorescent probe (Bright, 1988). The presence of absorbing components in solution that absorb light at or near the excitation wavelength of the fluorophore will have the effect of decreasing luminescence. In addition, noncovalent interactions of the probe with other components in solution can inhibit rotational freedom and quench fluorescence. 

Steady state anisotropy measurements described in Section 2.7.5 can sometimes be misleading if a number of sources of depolarization are present. For example, as discussed, it can be difficult to differentiate global molecular rotation from local rotational freedom of the fluorophore when it is attached to a larger molecule [ 1,127]. In these cases time resolved single molecule fluorescence anisotropy measurements can be of use since the contributions to the depolarization may well act on different timescales. For example, for a labelled protein segmental motion of the protein backbone near the label and motion in the linker by which... 

Fluorescence anisotropy can be correlated with the rotational freedom of the fluorophores present in the sample. High anisotropy values correspond to a low depolarization of the fluorescence emitted light and are indicative of the presence of fluorophores with low rotational freedom. In contrast, low anisotropy values are a consequence of a high depolarization of the emitted light and are commonly ascribed to the presence of highly mobile fluorophores. 

Fluorescence anisotropy values for the fiuorescence of a fluorophore on a protein will depend on the fluorophore s rotational freedom and fiuorescence lifetime. Because the motional freedom of intrinsic or extrinsic fluorophores will usually increase when a protein unfolds, a change in a protein s fluorescence anisotropy is expected upon unfolding. However, to properly use anisotropy to analyze the thermodynamics (or kinetics) of an unfolding transition, Eq. (1) should be replaced with one that includes the fluorescence quantum yield of the protein s structural states (see Reference 19). 

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