Frequency dependent anisotropy decay

Instead of recording separately the decays of the two polarized components, we measure the differential polarized phase angle A (co) = — i between these two components and the polarized modulation ratio A (co) = mfm . It is interesting to define the frequency-dependent anisotropy as follows ... 

The measurements are different in the frequency-domain. In this case we measure the phase shift between the parallel and perpendicular components of the emission, and a frequency-dependent anisotropy, which is analogous to the steady state anisotropy. These two types of data are used to determine the decay law for the anisotropy (equation 15). 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

By taking AC susceptibility of the complex over 1-1500 Hz from 1.8 to 3K, the out-of-phase susceptibility displays frequency dependence (Figure 9.26), however, none of the curves reaches a peak at 1.8 K. The DC magnetization decay method (Figure 9.26) [113, 114] determined effective barrier to be 18.4 K and a relaxation time constant of 2 x 10 s. As discussed above, Gd + is a pure spin ion the major anisotropy contribution is the Mn + ion, which is the most well known anisotropy source in the design of SMM and SCM. 

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. 

To illustrate the nature of the anisotropy decays the equivalent time-dependent anisotropies are shown as an insert. These were calculated from the frequency-do-main data. For Sj Nuclease the plot of log r(t) versus time is mostly linear with a slope of (12 nsec) This is the portion of the anisotropy decay due to overall rotational diffusion of the protein. The rapid component in the nuclease anisotropy decay is seen only near the t = 0 origin. The anisotropy decay of melittin is much more rapid, which reflects the greater motional freedom of the tryptophan residue in this disordered polypeptide. Because of the segmental motions which depolarize the... 

Figure 11.12. Frequency dependent values of for DPH in DPPC vesicles. The inset shows the recovered anisotropy decay. From Ref. dO.

The wavelength-dependent shifts in the differential polarized phase curves can be understood in terms of the comrifautions of various rotations to the anisotropy decay. As reasoned in our discussion of Figures I2.S and 12.6, both the in-plane and out-of-plane rotations are expected to contribute when rg = 0.4. Hence, the data for 3SI- and 442-nm excitation represent a weighted average of Di and Di- For rg vaiues near -0.2, one expects the in-plane rotation to be dominant (Hguie 12.11). This effect can be seen in the data for 281-nm excitation, for which the maximum value of A (absolute value) is shifted toward higher modulation frequencies. Finally, for rg values near... 

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