Protein fluorescence spectral relaxation

The fluorescence behaviour of a fluorophore is also influenced by the solvent, especially the solvent polarity [308]. Moreover, when a molecule is excited the solvent molecules around it rearrange. Consequently, energy is transferred to the solvent, with the result that the emission spectrum is red-shifted. Solvent (or spectral) relaxation in water happens on the time scale of a few ps. However, the relaxation times in viscous solvents and in dye-protein constructs can be of the same order as the fluorescence lifetime. The measurement of the solvent relaxation can therefore be used to obtain information about the local environment of fluorescent molecules [485]. 

Observation of reorientational dynamics of dipolar groups surrounding the fluorophore in response to changes in the dipole moment of the fluorophore occurring upon electronic excitation. Such dynamics result in the appearance of spectral shifts with time,(1 ) in changes of fluorescence lifetime across the fluorescence spectrum,(7,32) and in a decrease in the observable effects of selective red-edge excitation.(1,24 33 34) The studies of these processes yield a very important parameter which characterizes dynamics in proteins— the reorientational dipolar relaxation time, xR. 

The dipole-dipole interactions of the fluorophore in the electronic excited state with the surrounding groups of atoms in the protein molecule or with solvent molecules give rise to considerable shifts of the fluorescence spectra during the relaxation process. These spectral shifts may be observed directly by time-resolved spectroscopic methods. They may be also studied by steady-state spectroscopic methods, but in this case additional data must be obtained by varying factors that affect the ratio between tf and xp. 

As shown above, the intrinsic fluorescence spectra of proteins as well as coenzyme groups and probes shift within very wide ranges depending on their environment. Since the main contribution to spectral shifts is from relaxational properties of the environment, the analysis of relaxation is the necessary first step in establishing correlations of protein structure with fluorescence spectra. Furthermore, the study of relaxation dynamics is a very important approach to the analysis of the fluctuation rates of the electrostatic field in proteins, which is of importance for the understanding of biocatalytic processes and charge transport. Here we will discuss briefly the most illustrative results obtained by the methods of molecular relaxation spectroscopy. 

There are substantial difficulties in the interpretation of temperature-dependent shifts of protein spectra because of the thermal lability of proteins and the possibility of temperature-dependent conformational transitions. Low-temperature studies in aqueous solutions revealed that for many of the proteins investigated the observed shifts of the fluorescence spectra within narrow temperature ranges were probably the result of cooperative conformational transitions, and not of relaxational shifts/100 1 Spectral shifts have also been observed for proteins in glass-forming solvents, 01) but here there arise difficulties associated with the possible effects of viscous solvents on the protein dynamics. 

The fluorescent probe 2,6-TNS and other similar aminonaphthalene derivatives (1,8-ANS, DNS) were considered to be indicators of the polarity of protein molecules, and they were assumed to be bound only to hydrophobic sites on the protein surface. The detection of considerable spectral shifts with red-edge excitation has shown that the reason for the observed short-wavelength location of the spectra of these probes when complexed to proteins is not the hydrophobicity of their environment (or, at least, not only this) but the absence of dipole-relaxational equilibrium on the nanosecond time scale. Therefore, liquid solvents with different polarities cannot be considered to simulate the environment of fluorescent probes in proteins. 

In this chapter results of the picosecond laser photolysis and transient spectral studies on the photoinduced electron transfer between tryptophan or tyrosine and flavins and the relaxation of the produced ion pair state in some flavoproteins are discussed. Moreover, the dynamics of quenching of tryptophan fluorescence in proteins is discussed on the basis of the equations derived by the present authors talcing into account the internal rotation of excited tryptophan which is undergoing the charge transfer interaction with a nearby quencher or energy transfer to an acceptor in proteins. The results of such studies could also help to understand primary processes of the biological photosynthetic reactions and photoreceptors, since both the photoinduced electron transfer and energy transfer phenomena between chromophores of proteins play essential roles in these systems. 

Fluorescence sensitivity of calcofluor to the medium is common to many fluorophores such as TNS (McClure and Edelman, 1966), Trp residues (Burstein et al. 1973) and flavin (Weber, 1950). However, the fluorescence emission maxima of the above fluorophores are also viscosity dependent. Thus, the solvent polarity scale is insufficient to describe the spectral properties of a fluorophore in a protein (case of Trp residues) or bound to a protein (case of TNS). In fact, when the fluorophore is sun ounded by a rigid or viscous environment, or when it is bound tightly to a protein, its fluorescence emission will be located at short wavelengths. In this case, the emission occurs from a non-relaxed state, and the spectrum obtained will be identical to that obtained when the emission occurs from a hydrophobic environment such as isobutanol. Therefore, emission of calcofluor on HSA may be the result of an emission from a hydrophobic binding site and/or a highly rigid binding site. 

The effects of solvent polarity are best imderstood by specific examples. To model the fluorescence emission of proteins we examine spectra for iV-acetyl-L-tryptophanamide (NATA). This molecule is analogous to tryptophan in proteins. It is a neutral molecule, and its emission is more homogeneous than that of tryptophan itself. In solvents of increasing polarity the emission spectra shift towards longer wavelengths (Fig. 5). The emission maxima of NATA in dioxane, ethanol and water are 333, 344 and 357, respectively. These solvents are non-viscous, so the emission is dominantly from the relaxed state (Fig. 4). The spectral shifts can be used to calculate the change in dipole moment which occurs upon excitation [6]. More typically, the emission spectrum for a sample is compared with that foimd for the same fluorophore in various solvents, and the environment judged as polar or non-polar. While this approach is qualitative, it is simple and reliable, and does not involve the use of theoretical models or complex calculations. 

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