Tryptophan fluorescence depolarization

T. Ichiye and M. Karplus, Fluorescence depolarization of tryptophan residues in proteins A molecular dynamics study, Biochemistry 22, 2884-2894 (1983). 

Nishimoto, E., Yamashita, S., Szabo, A. G., and Imoto, T. (1998) Internal motion of lyzozyme studied by time-resolved fluorescence depolarization of tryptophan residues, Biochemistry 37, 5599-5607. 

Fluorescence depolarization measurements of aromatic residues and other probes in proteins can provide information on the amplitudes and time scales of motions in the picosecond-to-nanosecond range. As for NMR relaxation, the parameters of interest are related to time correlation functions whose decay is determined by reorientation of certain vectors associated with the probe (i.e., vectors between nuclei for NMR relaxation and transition moment vectors for fluorescence depolarization). Because the contributions of the various types of motions to the NMR relaxation rates depend on the Fourier transform of the appropriate correlation functions, it is difficult to obtain a unique result from the measurements. As described above, most experimental estimates of the time scales and magnitudes of the motions generally depend on the particular choice of model used for their interpretation. Fluorescence depolarization, although more limited in the sense that only a few protein residues (i.e., tryptophans and tyrosines) can be studied with present techniques, has the distinct advantage that the measured quantity is directly related to the decay of the correlation function. 

The study of fluorescence depolarization of tryptophans in proteins is likely to develop into a powerful tool for analyzing the internal motions.4593 Clarification of the photophysics of tryptophans by jet measurements,460 extension of the time scale of observation into the picosecond and femtosecond range,461 and utilization of normal or modified proteins with only a single tryptophan462 should lead to a significant increase in the utility of this experimental approach. 

Lakowicz and Maliwal, 1983, Oxygen quenching and fluorescence depolarization of tyrosine residues in proteins. Journal of Biological Chemistry 258,4794-4801. Lakowicz, J. R., Maliwal, B., Cherek, H. and Balter, A. 1983, Rotational freedom of tryptophan residues in proteins and peptides. Biochemistry. 22, 1741 - 1752. 

We have consistently talked about the polarization of a fluor bound to a macromoleeule. What about the intrinsic fluorescence of the macromolecules and its polarization For example the intrinsic fluorescence of tryptophan in a protein and utilization of its polarization in stud3dng the protein. There is a problem here. Large proteins move very slowly on a molecular scale. Thus, to observe depolarization due to motion, the lifetime of the excited state should be sufficiently long, i.e., there should be a good time lag between excitation and emission so that the molecule may show substantial movement in that time and depolarization may occur. For very small proteins, intrinsic fluorescence may be of some use, but for larger proteins, extrinsic fluorescence has to be made use of. 

Room temperature phosphorescence can be observed from dried proteins. Sheep wool keratin(47) has a phosphorescence lifetime of 1.4 s. Six lyophilized proteins were shown to exhibit phosphorescence at room temperature.(48) The spectra were diffuse, and the lifetime was non-single-exponential, which the authors interpreted as due to inhomogeneous distribution of tryptophans. As the protein was hydrated, the phosphorescence lifetime decreased. This decrease occurred over the same range of hydration where the tryptophan fluorescence becomes depolarized. Hence, these results are consistent with the idea that rigidity of the site contributes to the lifetimes. 

For chromophores that are part of small molecules, or that are located flexibly on large molecules, the depolarization is complete—i.e., P = 0. A protein of Mr = 25 kDa, however, has a rotational diffusion coefficient such that only limited rotation occurs before emission of fluorescence and only partial depolarization occurs, measured as 1 > P > 0. The depolarization can therefore provide access to the rotational diffusion coefficient and hence the asymmetry and/or degree of expansion of the protein molecule, its state of association, and its major conformational changes. This holds provided that the chromo-phore is firmly bound within the protein and not able to rotate independently. Chromophores can be either intrinsic—e.g., tryptophan—or extrinsic covalently bound fluorophores—e.g., the dansyl (5-dimethylamino-1-naphthalenesulfonyl) group. More detailed information can be obtained from time-resolved measurements of depolarization, in which the kinetics of rotation, rather than the average degree of rotation, are measured. For further details, see Lakowicz (1983) and Campbell and Dwek (1984). 

Researches on theoretical topics have not been reported very extensively. A few papers are mentioned here and some others at appropriate points later in the article. Weber has re-examined the famous Perrin equation for quantifying the rotational depolarization of fluorescence. The arguments presented in the paper are applied to the temperature dependence of the local motions of tyrosine and tryptophan residues observed in proteins. 

It is very important that the lifetime of the fluorophore (either intrinsic or extrinsic) being used to measure the anisotropy decay be appropriate to the timescale of the motions under examination. Too short a lifetime will mean that the fluorescence intensity will die away before much rotational motion takes place the anisotropy will remain high over the course of the fluorescence intensity decay and the latter s components will contain limited information about the motional dynamics. On the other hand, too long a lifetime means that the fluorescence will become too quickly depolarized on the timescale of the intensity decay, so that again the signal will contain limited information about the d3mamics. Generally, tryptophan fluorescence (with a lifetime of a couple of nanoseconds) can be reliably used to measure 4> values of up to about 25 ns. 

Proteins are generally excited at 295 nm in order to avoid (i) energy transfer from tyrosine to tryptophan residues, which can occur at shorter wavelengths (e.g. at 280 nm where tryptophan has its maximum absorbance) and which depolarizes the fluorescence, and (ii) depolarization of tryptophan fluorescence due to relaxation from its to electronic states. (At shorter wavelengths electrons are excited to both levels, after vdiich electrons in the hi er level relax radiationlessly to the lower, which also depolarizes the fluorescence.) One would like to maximize the initial anisotropy in order to obtain the most sensitivity for polarized fluorescence measurements. 

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