Associated Anisotropy Decay

Multiexponential anisotropy decay can also occur for a mixture of independendy rotating fluorophaes. As a example, such anisotropy deci can occur for a fluoiD-phore when some of the ftoorophoce midecriles ate boiind to protein and some are free in solution. The anisotropy from the mixture is an intensity-weighted average of contribution from the probe in eac] environment. 

Such systems can yield unusual anismropy ilecays which show miiuma and increases in aisotropies at long limes. Associated anisotropy rlecrys are described in morodelrHl in Chapter 12. 

Considerable attention has been given to the molecular interpretation of the limiting anisotropies (rj). The impetus for the analysis arises ffom a desire to understand the properties of the membranes that are responsible for the hind ed rotation. In one analysis,the rodlike DPH molecule is assumed to exist in a square-well potential such that its rotation is unhindered until acertain angle (Qc) is reached Rotation beyond this angle is as imed to be 

Hgure 11.10. Anisotropy decays of DPH in DPPC ve ks at 49.5 C, coniaming 0, and 50 mol % cholesteroL At 49.5 C the MW membranes are above their phase-transition temperature. M iich is near 37 Revised from Ref. 29. 

While this interpretation of the r- values is intuitively pleasing, the values of 9c should be int reted with caution. One difficulty of this model is that the calculation of 9c ff om rWro depends upon the existmee of a square-wdl 

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H. Szmacinski, R. Jayaweera, H. Cherek, and J. R. Lakowicz, Demonstration of an associated anisotropy decay by frequency-domain fluorometry, Biophys. Chem. 27, 233-241 (1987). 

There has been considerable interest in using fluorescence anisotropy to detect multiple environments in membranes as with fluorescence lifetimes (see above). For example, if a fluorophore is located in two environments with long and short lifetimes, then the fluorescence anisotropy decay process at longer times after excitation will be dominated by the long-lived fluorescent species. This occurs with parinaric acids, and this situation has been explored for a number of theoretical cases. 60 A similar situation has been found for DPH in two-phase lipid systems by collecting anisotropy decay-associated spectra at early and late times after excitation. 61 Evidence was found for more than one rotational environment in vesicles of a single lipid of it is at the phase transition temperature. It is important to identify systems showing associated anisotropy decays with more than one correlation time, each of... 

Also in this chapter, as the final anisotropy topic, we describe associated anisotropy decays. Such decays occur when the solution contaix more than one t pe of fluoro-phore ex the same fluorophcxe in different environments. Such sterns can r ult in complex anisotropy decays, even if all the individual species each di lay a single conelation time. 

C. Frequenq -E)oinain Measurements of Associated Anisotropy Decays... 

Calculation of an Associated Anisotropy Decay Use the intensity and anisotropy decays in Figure 12.25 to calcu-l c the anisotropy at 0,1, and 5 ns. Also calculate the anisoiropy values at 0,1, and 5 ns assuming a nonassoci-ated anisotropy decay ... 

Lee, J., Wang, Y., and Gibson, B. G. (1990). Recovery of components of fluorescence spectra of mixtures by intensity- and anisotropy decay-associated analysis the bacterial luciferase intermediates. Anal. Biochem. 185 220-229. 

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)... 

L. Davenport, J. R. Knutson, and L. Brand, Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. l,6-Diphenyl-l,3,5-hexatriene in lipid bilayers, Biochemistry 25, 1811-1816 (1986). 

As we explained in the previous section, fluorescence decays do not bring any direct evidence about energy transfer among DNA bases within a helix. In contrast, fluorescence anisotropy decays can provide this type of information. Such a possibility is based on the correlation of macroscopic observables to molecular parameters. On the molecular scale, r is related to the angle 6 formed between the transition dipoles associated to photon absorption and photon emission ... 

FPA results obtained at different salt conditions may not be directly comparable because the fluorescence properties of 6-MI, including the lifetime (t), are salt dependent. The salt dependence of the FPA of a helix in a complex construct should thereby be normalized relative to the FPA of a short control duplex of the same sequence of the targeted helix to account for salt effects on the local environment of the 6-MI fluorophore. The normalization ratio, rnoml, can be calculated as the ratio between the apparent rotational correlation time, 9, of the constructs and the control duplex only, rnomi = construct/ control- is related to the rate of anisotropy decay, with larger 9 associated with higher anisotropy. If the basic Perrin equation for a sphere (Eq. (14.3)) is used to simplify calculation, then... 

The evolution of the experimental anisotropy as a function of the temperature is shown in Fig. 8. As expected, the decay rate increases as the temperature increases. For the highest temperature (t > 50 °C), it can be noticed that the anisotropy decays from a value close to the fundamental anisotropy of DMA to almost zero in the time window of the experiment (about 60 ns). This means that the initial orientation of a backbone segment is almost completely lost within this time. This possibiUty to directly check the amplitude of motions associated with the involved relaxation is a very useful advantage of FAD. In particular, it indicates that in the temperature range 50 °C 80 °C, we sample continuously and almost completely the elementary brownian motion in polymer melts. Processes too fast to be observed by this technique involve only very small angles of rotation and cannot be associated with backbone rearrangements. On the other hand, the processes too slow to be sampled concern only a very low residual orientational correlation, i.e. they are important only on a scale much larger than the size of conformational jumps. 

From time-resolved fluorescence depolarization measurements, the anisotropy decay times (0) and the associated anisotropy ([>) have been determined for all first generation dendrimers using Eq.(l) ... 

From fluorescence depolarization measurements, anisotropy relaxation times and the associated anisotropy values have been determined for p-C2P1 p-C2P2, p-C2P3, and / -C2P. For the dendrimers with more than one chromophore, a two-exponen-tial function was found to be necessary to fit the experimental anisotropy decay traces (Table 1.2). The multichromophoric dendrimers present two-exponential decays in the anisotropy traces. The fast component (410 ps to 280 ps) of the anisotropy decay (Table 1.2) is found to decrease from p-C2P2 to p-C2P4. Contrary to the meta-substituted dendrimers m-C 1 P , the sum of the / , is now always close to the limiting value of the anisotropy even if 11 is larger than one. 

Measurement of the correlation time, and provided the viscosity of the medium is known, allows the determination of the hydrodynamic volume, hence the size of the particle where the fluorophore is embedded. This may in turn reflect an association process. For non-spherical particles, the anisotropy decay is given by more complex relations [24]. A time-dependent anisotropy may also indicate intramolecular mobility. 

We should note that, given the difference in quantum yield between the free and bound probe, the fractional intensities utilized in Fig. 7 actually represent small percentages of bound probe on a molar basis. In fact, considering the accuracy of the differential phase measurement (better than O.r) one can detect, in this system, on the order of 0.1% bound probe. This phenomenon also occurs in time-domain measurements. Specifically, if one monitors the anisotropy decay of a system which displays multiple lifetimes associated with multiple rotational diffusion rates then one may observe a decline at short times of the anisotropy followed by a rise at latter times and subsequent decrease. This dip and rise effect has been observed by Millar and co-workers in studies on protein-DNA interactions, specifically in the case of the interaction of a fluorescent DNA duplex with the Klenow fragment of DNA polymerase. 

Quenching studies of protein fluorescence provide answers regarding the accessibility of certain internal or external groups to quencher molecules. Another application concerns the study of associative behavior and properties of proteins and membranes. The rationale is that the fluorescence transition is polarized and this polarization can be exploited in time-resolved analysis and interpreted in terms of the rotation or tumbling motion which in turn is determined by the viscosity and structure of the environment of the fluorescing group. In particular, anisotropy decay studies have yielded a great deal of information on the mobility of natural and artificial membranes and/or the dynamics of proteins as well as small molecules in membranes. For such studies fluorescence lifetime labels that can be attached to proteins or that dissolve in membranes have... 

A second example of the need for lime-resolved measurements is the conq lex anisotropy decay displayed by a protein which self-associates into a tetramer Figure 4.5). Rotational motions can be measured from the decays of anisotropy. For a spherical molecule one expects a single decay time for the anisotropy, which is called the rotational correlati(Hi time (6). 

It is useful to compare the equations describing associated and nonassociated anisotropy decays. For a smgle fluoro-phore which displays multiexponential intensity and anisotropy decays, the parallel and perpendicular components of the emission are given by... 

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