The complexes cis-[Ru(phen)(Apy)], Apy = 4-aminopyridine and 3,4-aminopyridine, are stable in aqueous solution with strong visible absorption. They present emission in the visible region with long lifetime that accumulates in the cytoplasm of Neuro2A cell line without appreciable cytotoxicity. The complexes also serve as mixed-type reversible inhibitors of human AChE and BuChE with high active site contact. cis-[Ru(phen)(3,4Apy)] competes efficiently with DMPO by the OH radical. Luminescence using fluorescence lifetime imaging (FLIM) enables real-time imaging of the conformational changes of the self-aggregation of Aβ with incubation of complexes (0-24 h) in phosphate buffer at micromolar concentrations. By this technique, we identified protofibrills in the self-assembly of Aβ and globular structures in the short fragment Aβ in aqueous solution.
Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.
Static and time-resolved fluorescence of tryptophan and ortho-aminobenzoic acid was used to investigate the interaction of the synthetic antimicrobial peptide L1A (IDGLKAIWKKVADLLKNT-NH2) with POPC and POPC:POPG. N-acetylated (Ac-L1A) and N-terminus covalently bonded ortho-aminobenzoic acid (Abz-L1A-W8V) were also used. Static fluorescence and quenching by acrylamide showed that the peptides adsorption to the lipid bilayers was accompanied by spectral blue shift and by a decrease in fluorescence quenching, indicating that the peptides moved to a less polar environment probably buried in the lipidic phase of the vesicles. These results also suggest that the loss of the N-terminus charge allowed deeper fluorophore insertion in the bilayer. Despite the local character of spectroscopic information, conclusions can be drawn about the peptides as a whole. The dynamic behaviors of the peptides are such that the mean intensity lifetimes, the long correlation time and the residual anisotropy at long times increased when the peptides adsorb in lipid vesicles, being larger in anionic vesicles. From the steady-state increase in fluorescence intensity and anisotropy, we observed that the partition coefficient of peptides L1A and its Abz analog in both types of vesicles are higher than the acetylated analog; moreover, the affinity to the anionic vesicle is higher than to the zwitterionic.
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