Secondary Structure Transitions for a Family of Amyloidogenic, Antimicrobial Uperin 3 Peptides in Contact with Sodium Dodecyl Sulfate.

Secondary structure changes are an inherent part of antimicrobial (AMP) and amyloidogenic peptide activity, especially in close proximity to membranes, and impact the peptides' function and dysfunction roles. The formation, and stability of α-helical components are regarded as essential 'intermediates' for both these functions. To illuminate the conformational transitions leading to amyloid formation we use short cationic AMPs, from an Australian toadlet, Uperoleia mjobergii, (Uperin 3 family, U3) and assess the impact on secondary structural elements in the presence of a membrane mimetic surfactant, sodium dodecyl sulfate (SDS). Specifically, Uperin 3.x, where x=4, 5, 6 wild-type peptides and position seven variants for each, R7A or K7A, were investigated using a combination of experimental and simulation approaches. In water, U3 peptides remain largely unstructured as random coils, with the addition of salts initiating structural transitions leading to assembly towards amyloid. Solution NMR data show that an unstructured U3.5 wt peptide transitions in the presence of SDS to a well-defined α-helical structure that spans nearly the entire sequence. Circular dichroism (CD) and ThT fluorescence studies show that all six U3 peptides aggregate in solution, albeit with vastly varying rates, and a dynamic equilibrium between soluble aggregates rich in either α-helices or ß-sheets may exist in solution. However, the addition of SDS leads to a rapid disaggregation for all peptides and stabilisation of predominantly α-helical content in all the U3 peptides. Molecular dynamics (MD) simulations show that the adsorption of U3.5 wt/R7A peptides onto the SDS micelle is driven by Coulombic attraction between peptide cationic residues and the negatively charged sulfate head-groups on SDS. Simulating the interactions of various kinds of ß-sheet dimers (of both U3.5 wt and its variant U3.5 R7A) with SDS micelles confirmed ß-sheet content decreases in the dimers after their attachment to the SDS micelle. Adsorbed peptides interact favourably with the hydrophobic core of the micelle, promoting intramolecular hydrogen bonds leading to stabilisation of the α-helical structure in peptides, and resulting in a corresponding decrease in intermolecular hydrogen bonds responsible for ß-sheets.

Sequence comparisons of cytochrome P450 aromatases from Australian animals predict differences in enzymatic activity and/or efficiency†.

Aromatase (P450arom, CYP19A1) is the terminal enzyme in the synthesis of the steroid hormone family of estrogens. Not surprisingly, this enzyme has structural similarities between the limited number of species studied thus far. This study examined the structure of aromatases from four diverse Australian species including a marsupial (tammar wallaby; Macropus eugenii), monotreme (platypus; Ornithorhynchus anatinus), ratite (emu; Dromaius novaehollandiae) and lizard (bearded dragon; Pogona vitticeps). We successfully built homology models for each species, using the only crystallographically determined structure available, human aromatase. The amino acid sequences showed high amino acid sequence identity to the human aromatase: wallaby 81%, platypus 73%, emu 75% and bearded dragon at 74%. The overall structure was highly conserved among the five species, although there were non-secondary structures (loops and bends) that were variable and flexible that may result in some differences in catalytic activity. At the N-terminal regions, there were deletions and variations that suggest that functional distinctions may be found. We found that the active sites of all these proteins were identical, except for a slight variation in the emu. The electrostatic potential across the surfaces of these aromatases highlighted likely variations to the protein-protein interactions of these enzymes with both redox partner cytochrome P450 reductase and possibly homodimerization in the case of the platypus, which has been postulated for the human aromatase enzyme. Given the high natural selection pressures on reproductive strategies, the relatively high degree of conservation of aromatase sequence and structure across species suggests that there is biochemically very little scope for changes to have evolved without the loss of enzyme activity.