The local environment and dynamics of the single tryptophan residue in the respective active loops of cardiotoxin and alpha-neurotoxin from Naja nigricollis and of erabutoxin b from Laticauda semifasciata have been studied by steady-state and time-resolved polarized fluorescence and analyzed with distributions of decay times. Trp11 in loop I of cardiotoxin exhibits a very broad and complex distribution of fluorescence lifetimes at 20 degrees C. Despite its relatively external location in the toxin, the residue appears to be partly shielded from water and shows restricted but significant conformational fluctuations on the picosecond and nanosecond time scales. The thermal stability of cardiotoxin allowed a study of its static and dynamic fluorescence properties over a large range of temperatures. Interconversions in the intermediate nanosecond range lead to a thorough reorganization of the cardiotoxin fluorescence lifetime distribution with temperature. On the contrary, the fluorescence kinetics of Trp29 in loop II of the two neurotoxins is dominated by about 80% of a major decay time, which suggests that a nearly unique local conformation of the toxin is maintained over all time scales above the sub-nanosecond range. The fluorescence anisotropy decays show that the residue also has extremely limited rotational freedom down to the picosecond time scale. These findings are in good agreement with structural and dynamic information previously reported on the different toxins from NMR and X-ray crystallographic studies. The different dynamic properties around the tryptophan residue of the cardiotoxin and neurotoxin active loops can be analyzed within the frame of their different respective mechanisms of toxicity.

A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two tryptophan residues in an unfolded form of yeast phosphoglycerate kinase [Garcia, P., et al. (1995) Biochemistry 34, 397-404]. In this paper, we present a more detailed characterization of these residual structures, through the study of two single tryptophan-containing mutants of yPGK, W333F and W308Y. Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures. On the other hand, the compactness and dynamics of the protein matrix were probed using tryptophan fluorescence quenching by acrylamide at various denaturant concentrations. Taking into consideration the changes in sample viscosity induced by addition of guanidinium chloride made feasible the use of this technique during the denaturation process. These different approaches have shown that the residual structures around tryptophan 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding tryptophan 333. Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy. In the native protein, tryptophan buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas tryptophan that is partially accessible to the solvent, W308, has a lifetime close to 0. 5 ns. The time-resolved tryptophan fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single tryptophan mutants. The significance of minor long lifetime components is discussed for the two tryptophan residues. This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of tryptophan 333 is modified for denaturant concentrations lower than those for tryptophan 308.

After purification to homogeneity by Bio-Rex 70 ion exchange chromatography, micromolar solutions ofNaja nigricollis cardiotoxin were found to contain significant amounts of aggregates, as detected by time-resolved polarized fluorescence of its single tryptophan residue. The level of cardiotoxin aggregation depends strongly and reversibly on the protein concentration and pH. However, supplementary reverse-phase HPLC completely suppresses this aggregation, resulting in all cases in fluorescence anisotropy decays characteristic of the pure cardiotoxin monomer. The self-association properties of cardiotoxin, in the presence of a possible cofactor eliminated by the HPLC step, may be functionally relevant, and would deserve further investigation. The physical heterogeneity of the cardiotoxin samples required an appropriate model for the analysis of fluorescence depolarization, which was iteratively improved by comparison with experimental results. In this way, an approximate molar fraction of 10-15% aggregated cardiotoxin at a 90ΜM total protein concentration, pH 7, was determined. The fluorescence of the partly aggregated samples is significantly perturbed as compared to the HPLC-treated monomer, indicating that the cardiotoxin aggregate must have an increased average fluorescence lifetime and a strongly decreased initial anisotropy. The decrease in initial anisotropy suggests either an increased mobility of the tryptophan residue upon aggregation or fast energy transfers between residues of different cardiotoxin molecules brought within a short distance in the aggregate. This study illustrates the high sensitivity of the time-resolved fluorescence technique, through both total fluorescence and anisotropy parameters, to low levels of physical or chemical heterogeneity in a protein sample.

Violacein and deoxyviolacein are bis-indole pigments synthesized by a number of microorganisms. The present study describes the biosynthesis of a mixture of violacein and deoxyviolacein using a genetically modified Y. lipolytica strain as a production chassis, the subsequent extraction of the intracellular pigments, and ultimately their purification using column chromatography. The results show that the optimal separation between the pigments occurs using an ethyl acetate/cyclohexane mixture with different ratios, first 65:35 until both pigments were clearly visible and distinguishable, then 40:60 to create a noticeable separation between them and recover the deoxyviolacein, and finally 80:20, which allows the recovery of the violacein. The purified pigments were then analyzed by thin-layer chromatography and nuclear magnetic resonance.