Proteins fluorescence depolarization

J. R. Lakowicz and B. Maliwal, Oxygen quenching and fluorescence depolarization of tyrosine residues in proteins, J. Biol. Chem. 258, 4794-4801 (1983). 

Figure 2.4. Schematic representation of processes which lead to fluorescence depolarization in proteins rotation of the protein molecule as a whole with correlation time rotation of the fluorophore with correlation time d, and excitation energy transfer, represented by the wavy arrow.

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

The nmr data for this type of motion are direct and the motion clearly involves rotation about bonds in the millisecond time scale range. However. less direct evidence for motion comes from other techniques such as fluorescence depolarization, 02 diffusion, hydrogen exchange kinetics, and nmr relaxation times (see Ref. 4). The extent of this motion is not yet easy to define, but this evidence points to motion in the nanosecond time scale range. It is tempting to see the motion in this time scale as bond oscillations rather than rotations. To put it in a different way, on this time scale the side chains have some freedom to move with respect to each other but not normally to undergo substantial bond rotation. Table IV summarizes some references for motion of different types. Additionally, nmr relaxation studies suggest that the backbone or main chain of a protein is more restricted than that of the side chains. 

We have not measured fluorescence depolarization with fluorophors and our polymers, but such measurements have been made by others, particularly with proteins and, as you indicate, it is possible to determine rotational relaxation times for the macromolecule and thus to obtain some insight into its behavior in solution. 

Royer, C.A., Tauc, P., Herve, G. and Brochon, J.-C. (1987). Ligand binding and protein dynamics a fluorescence depolarization study of aspartate transcarbamylase from Escherichia coli. Biochemistry, 26, 6472-6478. 

Secondly, the technique of fluorescent depolarization which was introduced into protein chemistry by Weber in 1952... 

The technique of fluorescence depolarization uses in principle fluorescent dyes covalently bound to proteins. The label is excited by polarized light the fluorescence is also polarized to a degree that is inversely related to the amount of Brownian motion occurring during the interval between absorption and emission of the light. The information about the amount of motion helps to form an idea on the size and shape of the protein being investigated. 

Here, we discuss the polarization of fluorescence emission. The spatial orientations of emitting fluorophores determine the polarization of photons emitted. This relationship is the basis of fluorescence depolarization experiments as illustrated in Fig. 4a. When a sample of randomly oriented molecules (e.g., proteins... 

Similar to fluorescence depolarization and NMR, two limiting cases exist in which the molecular motion becomes too slow or too fast to further effect the ESR lineshape (Fig. 8) (35). At the fast motion limit, one can observe a narrow triplet centered around the average g value igxx + gyy + giz with a distance between lines of aiso = Axx- -Ayy- -A2,z)l3, where gu and Ajj are principal values of the g-tensor and the hyperflne splitting tensor A, respectively. At the slow motion limit, which is also referred to as the rigid limit, the spectrum (shown in Fig. 8) is a simple superposition of spectra for all possible spatial orientations of the nitroxide with no evidence of any motional effects. Between these limits, the analysis of the ESR lineshape and spectral simulations, which are based on the Stochastic Liouville Equation, provide ample information on lipid/protein dynamics and ordering in the membrane (36). 

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. 

Table 1.4 Rotational correlation times x, of proteins in aqueous solution at 25 °C determined by time-resolved fluorescence depolarization measurements [37].

The test that has gained widest acceptance is the determination of the amniotic fluid lecithin/sphingomyelin ratio, using thin-layer chromatography (TLC). By its nature, this is a time-consuming and labor-intensive test, and an alternative based upon fluorescence depolarization has been proposed and widely adopted. This procedure measures the ratio of surfactant to protein in amniotic fluid. 

Evidence for mobility within proteins comes from a variety of physical methods single crystal X-ray or neutron diffraction, electron microscopy, and spectroscopic techniques such as NMR, fluorescence depolarization, Mossbauer spectroscopy and H-exchange studies. Theoretical approaches such as potential-energy minimization and molecular-dynamics calculations may also be used to study flexibility. An illustration of the frequency range of the various thermal motions detected in proteins is given in Table 1. 

The dynamics of proteins occur on many time scales They extend from subpicosecond instantaneous relaxation processes after electronic excitations reorientation of residues in the time regime from 10-100 ps as measured by fluorescence depolarization up to denaturation and unfolding of proteins in the time regime of seconds ... 

In contrast, the term associated with the slow relaxation will show a strong field dependence and, depending on the value of the order parameter, will make a large contribution to T. As for fluorescence depolarization, simulations can provide reasonable estimates of NMR order parameters and fast relaxation times of proteins, but not the full picture of the relaxation. 

An important problem in the study of protein folding is the control of the initial state (i.e., obtaining as starting material a completely unfolded protein). Therefore a very careful characterization of the denatured protein is the first step of the experimental work the second step is finding good conditions for reversibility. Different methods can be used to characterize the unfolded proteins hydrodynamic methods such as viscosity and sedimentation coefficient determination spectroscopic methods (absorption and fluorescence), fluorescence depolarization, CD, ORD and optical rotation, and NMR. Chemical methods provide a more direct tool for measuring the accessibility... 

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