Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity.

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 A and T. cruzi at 1.9 A resolution, and that from P. falciparum at a higher resolution (2.0 A) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.

A micelle nucleation model for the interaction of dodecyl sulphate with Lys49-phospholipases A(2).

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of Bothrops jararacussu that lacks detectable catalytic activity, yet causes rapid Ca(2+)-independent membrane damage. With the aim of understanding the interaction between BthTx-I and amphiphilic molecules, we have studied the interaction of sodium dodecyl sulphate (SDS) with the protein. Circular dichroism and attenuated total reflection Fourier-transform infrared spectra of BthTx-I reveal changes in the alpha-helical organization of the protein at an SDS/BthTx-I molar ratio of 20-25. At SDS/BthTx-I ratios of 40-45 the alpha-helices return to a native-like conformation, although fluorescence emission anisotropy measurements of 2-amino-N-hexadecyl-benzamide (AHBA) demonstrate that the total SDS is below the critical micelle concentration when this transition occurs. These results may be interpreted as the result of SDS accumulation by the BthTx-I homodimer and the formation of a pre-micelle SDS/BthTx-I complex, which may subsequently be released from the protein surface as a free micelle. Similar changes in the alpha-helical organization of BthTx-I were observed in the presence of dipalmitoylphosphatidylcholine liposomes, suggesting that protein structure transitions coupled to organization changes of bound amphiphiles may play a role in the Ca(2+)-independent membrane damage by Lys49-PLA(2)s.

The interaction between heparin and Lys49 phospholipase A2 reveals the natural binding of heparin on the enzyme.

We have studied at a molecular level the interaction of heparins on bothropstoxin-I (BthTx-I), a phospholipase A2 toxin. The protein was monitored using gel filtration chromatography, dynamic light scattering (DLS), circular dichroism (CD), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. The elution profile of the protein presents a displacement of the protein peak to larger complexes when interacting with higher concentration of heparin. The DLS results shows two Rh at a molar ratio of 1, one to the distribution of the protein and the second for the action of heparin on BthTx-I structures, and a large distribution with the increase of protein. The interaction is accompanied by significant changes in the CD spectra, showing two common features: a decrease in signal at 208 nm (3 and 6 kDa heparins) and an isodichroic point near 226 nm (3 kDa heparin). FTIR spectra indicate that only a few amino acid residues are involved in this interaction. Alterations in the ITFE by binding heparins suggest that the initial binding occurs on the ventral face of BthTx-I. Together, these results add an experimental and structural basis on the action mechanism of the heparins over the phospholipases A2 and provide a molecular model to elucidate the interaction of the enzyme-heparin complex at a molecular level.

Photoacoustic spectroscopy of aromatic amino acids in proteins.

This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.

An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A2 and amphiphilic molecules.

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A2 from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios>10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios<8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios>10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant.

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A2 promoted by amphiphilic molecules.

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca(2+)-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca(2+) ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)s), substitution of the Asp49 by Lys results in loss of Ca(2+) binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA(2)s cause Ca(2+)-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca(2+)-independent membrane damaging activity by approximately 4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accompanied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s.

Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet.

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize ( Zea mays) and pearl millet ( Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high alpha-helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, (1)H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded alpha-helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.