Structure-function relationships of bolaamphiphilic peptides and peptide hybrids

[摘要] Synthetic peptides derived from the active core of a natural antimicrobial peptide were used asa template for the design of novel bolaamphiphilic peptides and hybrid molecules. The amphiphiliccharacter of the original compounds was modified by using non-natural amino acids (AAs) – suchas ω-AA – and varying the hydrophobic content. The outcomes of these modifications were studiedfocusing on structural and biological properties.Because of the bolaamphiphilic character, the alternation of polar and non-polar AAs and theuse of hydrophobic AAs such as tyrosine and leucine, these novel molecules were designed toundergo self-assembly in response to certain stimuli (e.g. a pH increase). This significant propertywas investigated by means of different tools, such as fluorescence measurements, electronmicroscopy (EM), Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD).By using fluorescence it was possible to determine the critical aggregation concentration (CAC) ofthe new compounds. Differences in amino acid composition, which were reflected into diversesecondary structures and hydrophobicity (H), resulted in different CAC values and aggregationprofiles. The data were consistent with the literature and showed that (i) the aggregation of thesebasic compounds was triggered by a pH increase, (ii) the use of hydrophobic AA highly augmentedthe self-assembly tendency while (iii) the presence of proline strongly reduced it. EM revealed themorphology of the peptide assemblies: microtubes and microvesicles were identified andcharacterised by dimensions of 500 nm to 2 μm. The presence of 3-way junctions and vesiclesbudding out of the microtubes demonstrated that the self-assembly is a dynamic process. Theaggregation was confirmed by FT-IR spectroscopy, by studying the dried peptide assemblies andthe significant spectral signs the process left, especially in the amide II envelope.The relationship between hydrophobicity and self-assembly was expanded by experimentallyand theoretically determining the hydrophobic content of the novel bolaamphiphiles. Data fromliquid chromatography and computational calculations (two common ways used to determine thehydrophobicity of a given molecule) correlated well with the tendency to self-assemble, asexpressed by CAC values. Importantly, some structural parameters (such as the presence of β-turninduced by proline) also showed significant influence on the aggregation, highly limiting the roleof the peptides' hydrophobicity.These novel peptide bolaamphiphiles displayed a very low haemolytic action and retainedsome antimicrobial activity at high concentrations against both Gram-positive and -negativebacteria. Unfortunately, the activity was greatly reduced at low concentrations, as clearlydemonstrated by the use of two antimicrobial tests. The inability to provoke cell lysis was alsoevident when using liposomes mimicking a negative bacterial membrane. The loss of activity is possibly related to the modifications of the three-dimensional structurecaused by the use of ω-AA and proline, which strongly alter the secondary structure.The results of this study were valuable in terms of understanding the relationships betweenself-assembly and structural parameters, such as AA compositions, hydrophobicity and secondarystructure. Possible applications of the synthesised compounds were however limited as a result ofthe loss of the biological activity at low concentrations.

[发布机构] Stellenbosch University