Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (AA), spectral maximum position (&), fluorescence quenching by external ionic quenchers, lifetime (T), and quantum yield (4) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (A,,, = 330-332 nm, AA = 48-49nm, 4 = 0.1 1, 7 = 2.1 ns) and two on the surface. One of the latter is completely exposed to water (A,,l = 350-353 nm, AA = 59-61 nm, q = 0.2, T = 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (A,lt = 340-342 nm, AA = 53-55 nm, q = 0.3, T = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence Lifetime for various proteins, show good concordance with independent experimentally determined values.

In our previous paper (Reshetnyak, Ya. K., and E. A. Burstein. 2001. Biophys. J. 81:1710-1734) we confirmed the existence of five statistically discrete classes of emitting tryptophan fluorophores in proteins. The differences in fluorescence properties of tryptophan residues of these five classes reflect differences in interactions of excited states of tryptophan fluorophores with their microenvironment in proteins. Here we present a system of describing physical and structural parameters of microenvironments of tryptophan residues based on analysis of atomic crystal structures of proteins. The application of multidimensional statistical methods of cluster and discriminant analyses for the set of microenvironment parameters of 137 tryptophan residues of 48 proteins with known three-dimensional structures allowed us to 1) demonstrate the discrete nature of ensembles of structural parameters of tryptophan residues in proteins; 2) assign spectral components obtained after decomposition of tryptophan fluorescence spectra to individual tryptophan residues; 3) find a correlation between spectroscopic and physico-structural features of the microenvironment; and 4) reveal differences in structural and physical parameters of the microenvironment of tryptophan residues belonging to various spectral classes.

The physical causes for wide variation of Stokes shift values in emission spectra of tryptophan fluorophores in proteins have been proposed in the model of discrete states (Burstein, E. A., N. S. Vedenkina, and M. N. Ivkova. 1973. Photochem. Photobiol. 18:263-279; Burstein, E. A. 1977a. Intrinsic Protein Luminescence (The Nature and Application). In Advances in Science and Technology (Itogi Nauki i Tekhniki), Biophysics Vol. 7. VINITI, Moscow [In Russian]; Burstein, E. A. 1983. Molecular Biology (Moscow) 17:455-467 [In Russian; English translation]). It was assumed that the existence of the five most probable spectral classes of emitting tryptophan residues and differences among the classes were analyzed in terms of various combinations of specific and universal interactions of excited fluorophores with their environment. The development of stable algorithms of decomposition of tryptophan fluorescence spectra into log-normal components gave us an opportunity to apply two mathematically different algorithms, SImple fitting with Mean-Square criterion (SIMS) and PHase-plot-based REsolving with Quenchers (PHREQ) for the decomposition of a representative set of emission spectra of proteins. Here we present the results of decomposition of tryptophan emission spectra of >100 different proteins, some in various structural states (native and denatured, in complexes with ions or organic ligands, in various pH-induced conformations, etc.). Analysis of the histograms of occurrence of >300 spectral log-normal components with various maximum positions confirmed the statistical discreteness of several states of emitting tryptophan fluorophores in proteins.

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The smooth fluorescence bands of various organic fluorophores of different classes (e. g. indole, tryptophan, tyrosine, phthalimides, quinine sulfate, aminopyridines, acetylanthracenes) in different ionization states of their substituents and dissolved in various solvents can be accurately fitted on the frequency (wavenumber) scale by the four‐parametric log‐normal distribution curves up to the far spectral wings. This fact suggests that the log‐normal function is a good analytical description for smooth emission spectra of so‐called complex molecules. Examination of log‐normal parameters of 126 spectra of several tryptophan derivatives measured in different ionization states in various solvents revealed a straight‐linear relation between three shape parameters of the log‐normal function, namely, the positions of spectral maxima and of the two half‐maximal amplitudes. This fact indicates that only one parameter is needed to describe the band shape for the set of tryptophan derivatives. A similar linearity was also observed for the series of spectra of 1‐ and 2‐acetylanthracenes or 3‐amino‐N‐methyl‐phthalimides when varying the solvent. As a result, the spectral maximum position can entirely determine the shape of emission spectra of a series of a fluorophore derivatives in any environmental condition. Such a simplified mathematical representation significantly facilitates the problem of analytical resolution of composite fluorescence spectra, for instance, those of proteins.