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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.

The calcium binding by parvalbumin of whiting (Gadus merlangus) has been studied using tryptophanyl fluorescence characteristics. Titration of Ca2+-free parvalbumin with Ca2+ leads to a very pronounced blue shift, narrowing and intensification of the fluorescence spectrum. These spectral changes proceed in two stages reflecting the existence of at least three forms which can be interpreted as (a) the protein without Ca2+, (b) with one Ca2+ and (c) with two bound Ca2+ ions/molecule. The fluorescence of these forms has been identified and the fluorescence spectra measured at varied Ca2+ concentrations were resolved into three components corresponding to these spectral forms.

The chloroplast outer membrane contains different, specialized pores that are involved in highly specific traffic processes from the cytosol into the chloroplast and vice versa. One representative member of these channels is the outer envelope protein 16 (OEP16), a cation-selective high conductance channel with high selectivity for amino acids. Here we study the mechanism and kinetics of the folding of this membrane protein by fluorescence and circular dichroism spectroscopy, using deletion mutants of the two single tryptophanes Trp-77-->Phe-77 and Trp-100-->Phe-100. In addition, the wild-type spectra were deconvoluted, depicting the individual contributions from each of the two tryptophan residues. The results show that both tryptophan residues are located in a completely different environment. The Trp-77 is deeply buried in the hydrophobic part of the protein, whereas the Trp-100 is partially solvent exposed. These results were further confirmed by studies of fluorescence quenching with I(-). The kinetics of the protein folding are studied by stopped flow fluorescence and circular dichroism measurements. The folding process depends highly on the detergent concentration and can be divided into an ultrafast phase (k > 1000 s(-1)), a fast phase (200-800 s(-1)), and a slow phase (25-70 s(-1)). The slow phase is absent in the W100F mutant. Secondary structure analysis and comparision with closely related proteins led to a new model of the structure of OEP16, suggesting that the protein is, in contrast to most other outer membrane proteins studied so far, purely alpha-helical, consisting of four transmembrane helices. Trp-77 is located in helix II, whereas the Trp-100 is located in the loop between helices II and III, close to the interface to helix III. We suggest that the first, very fast process corresponds to the formation of the helices, whereas the insertion of the helices into the detergent micelle and the correct folding of the II-III loop may be the later, rate-limiting steps of the folding process.

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