Anisotropy Decays of Proteins

In the fuevious section we saw that in the native state RNase Ti di lsys a single-eiqKinential anisotropy deesy. This was an unnsual r ult, as most proteins diow n id components in the anisotropy dec s due to s m tal 

Fast ccMT onents in the anisotn y decays are characteristic of many proteins and pq rides and have been reported in many publications. Picosecond-timescale motions were also reported for tyrosine residues in pro-teins in these studies, a streak camera was used to obtain adequate time resolution. The short correlation rime observed in protons is variable and ranges from 50 to 5(X) ps, with the values being detemuned in part by the rime resolution of the instrument. The shorty correlation time is )proximately equal to that observed for NAIA in w er or f M snoall peptides (Table 17.5). These shor correlation times are typically insensitive to protein folding and are not greatly affected ly the viscosity of the solution. 

Die picosecond con onent in the anisotropy decays of proteins does appear to display some characteristic features. The fraction of the total anisotrqiy due to this component is typically larger for small unstructured peptides than for tryptophan residues buried in a protein matrix. Unfolding of a protein usually increases the an li-tilde of the fast motions. 

For proteins it is tempting to assume that tryptophan residues exposed to the aqueous phase will show the most rafhd anisotropy decays. However, this is not always the case. As an example, we note the anisotropy decay of staphylococcal nuclease. Hiis protein contains a single tryptophan residue at position 140, which is on the surface of the prot and near the C-tenninus. One might expect this region of the protein to rotate freely. In spite of this locati m in a seemingly unhindered environment, the anisotropy of tip-140 decays predominantly with a correla- 

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J. R. Lakowicz, G. Laszko, I. Gryczynski, and H. Cherek, Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry, J. Biol. Chem. 261, 2240-2245 (1986). 

In addition to being affected by shape, anisotropy decays are affected by the segmental flexibility of the macromole-cule. For instance, tryj ophan anisotropy decays of proteins frequently display correlation times that are too short to be due to rotational diffusion of the whole macromole-cule. These short-coirelation-time components are often due to independent motions of the tryptophan residue within the protein. Measurements of these components... 

Borst JW, Flink MA, van Hoek A, Visser AJWG (2005) Effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins. JFluoresc 15 153-160... 

Volkmer, A., Subramaniam, V., Birch, D. J. and Jovin, T. M. (2000). One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys. J. 78, 1589-98. 

Lipid-protein interactions are of major importance in the structural and dynamic properties of biological membranes. Fluorescent probes can provide much information on these interactions. For example, van Paridon et al.a) used a synthetic derivative of phosphatidylinositol (PI) with a ris-parinaric acid (see formula in Figure 8.4) covalently linked on the sn-2 position for probing phospholipid vesicles and biological membranes. The emission anisotropy decays of this 2-parinaroyl-phosphatidylinositol (PPI) probe incorporated into vesicles consisting of phosphatidylcholine (PC) (with a fraction of 5 mol % of PI) and into acetylcholine receptor rich membranes from Torpedo marmorata are shown in Figure B8.3.1. 

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)... 

Bucci, E., and Steiner, R. F. (1988). Anisotropy decay of fluorescence as an experimental approach to protein dynamics. Biophys. Chem. 30, 199—224. 

If we measure the anisotropy decay of the Trp residue of the protein as a function of time, based on the answer you found in question 1, what would be the expected result ... 

Interesting applications of anisotropy decays for proteins often develop not from tumbling of the protein as a whole, but from other reorientational degrees of freedom. These motions may include protein domain motions or segmental motions in proteins and peptides. The anisotropy decay in this case is non-single-exponential (see Fig. 4c) and takes the form ... 

Figure 7.12 Femtosecond-resolved fluorescence anisotropy decays of HPMO in Dioxane, normal micelle, DM-j8-CD and HAS protein [58]. Note the effect of confinement on the rotational motion of the probe.

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. 

At present, there is widespread interest in directly measuring the time-dependent processes. This is because considerably more information is av lilable from the time-dependent data. For example, the time-dependent decays of protein fluorescence can occasionally be used to recover the emission spectra of individual tryptophan residues in a protein. The time-resolved anisotropies can reveal the shape of the protein and/or the extent of residue mobility within the protein. The time-resolved energy transfer can reveal not only the distance between the donor and acceptor, but also the distribution of these distances. The acquisition of such detailed information requires high resolution instrumentation and careful data acquisition and analysis. 

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... 

The theory for rotational diffusion of non-spherical particles is complex. In theory the anisotropy decay of such a molecule can be composed of a sum of up to five exponentials [134]. The ellipsoids of revolution represent a smooth and symmetrical figure, which is often used for the description of the hydrodynamic properties of proteins. They are three-dimensional bodies generated by rotating an ellipse about one of its characteristic axes. In this case the anisotropy decay displays... 

The anisotropy decay of LADH excited at 300 nm is shown in Ft gure 11.13. The decay was found to be a single exponential with a correlation time of 33 ns. This single correlation time can be compared with that predicted for a hydrated sphere (0.2 g of H20/g of protein) from Eq. [10.52]), which is 31 ns at 20 C. Hence, this tryptophan residue appears to be rigidly held within the protein matrix. 

We now describe the theory for the anisotropy decays of ellipsoids of levolution. It is important to remember that this theory assumes that the ellipsoid is rigid. In the case of a labeled protein, this assumption implies no independent motion of the fluorophore vrithin the protein. For prolate or oblate ellipsoids, the anisotropy decay is expected to display three conelation times. 

While the use of glc al analysis with quoiching is us ul for studies of rigid anisotropic rotors Ais method is even more useful in studies of complex anisotropy decays from proteins. This is because the correlation times for segmra-tal motions and overall rotations of a protein are usually quite different in magnitude, whereas the correlation dmes of a rigid rotor are usually similar in magnitude. 

EFFECTS OF PROTEIN STRUCTURE ON THE INTENSITY AND ANISOTROPY DECAY OF RIBONUCLEASE T,... 

Lakowicz, J. R.. Gryezynski, I., Szmacinski, H., CSierek, Hh and Joshi, N., 1991, Anisotropy decays of single tryptophan proteins measured by GHz frequency-dorasin fluorometry whh ooUisImial flenching, Eur. Biophys. J. 19 125-140. 

I 7 nwsphorescence anisotropy decay In ghosts. The anisotropy decay of eosin-labelled proteins was monitored as a function of time. The data were fitted according to eQuaUon 21. Reivinted in part with permission from Mateyoshi, E. D. and Jovin, T. M. (1991) Biochemistry, 30, 3527-3538. Copyright 1991 American Chemical Society. 

Benci, S. Bottiroli, G. Schianchi, G. Vaccari, S. Vaghi, P. Luminescence and anisotropy decays of N-3-pyrene maleimide labeling IgG proteins and cells. J. Fluoresc. 1993, 3, 223-227. 

In order to avoid complications caused by excitation energy transfer between tryptophan residues, most investigations have been performed with proteins containing one tryptophan residue per molecule. When studying protein solutions, there are difficulties in separating the effects of rotation of entire protein molecules and of the chromophores themselves relative to their environment in the protein matrix. It is usually assumed that intramolecular motions are more rapid and manifest themselves as short-lived components of anisotropy decay curves or in depolarization at short emission lifetimes. 

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