Effect of peptide concentration on water structure, morphology, and thermal stability of self-assembling (rada)₄ peptide matrices

In order to elucidate design principles for peptide-based materials, we investigate (RADA)₄ oligopeptides that self-assemble to form β-sheet rich nanofibers that further develop into a viscoelastic 3D matrix in aqueous solution. Despite the fact that these materials can form hydrogels that are up to 99.5% (w/v) water, there is a limited understanding of water structure within these matrices. (RADA)₄nanofibers were formed in solutions at various temperatures (25 to 80 °C, and cooled to 25 °C) and peptide concentrations (0.5-3.0% (w/v)), and characterized using Circular Dichroism (CD), Transmission Electron Microscopy (TEM) and Differential Scanning Calorimetry (DSC). CD and TEM results showed some contradictions, where CD data suggested no nanofiber had formed but TEM images clearly showed nanofibers present. These results call into question previously published results where CD has been used solely for proving nanofiber formation. It was found that secondary structure of assembled peptides was affected by peptide concentration. Of significant interest was the structure of the water within the self-assembled structures. Where it was observed using DSC that the bound water structure was dramatically affected by peptide concentration. The unexpected results showed that at 3.0% (w/v) of peptide the amount of non-frozen water approached 80%, perhaps reflecting the synergy between closely packed nanofiber surfaces in interacting with water. However, it could be that the bound water content may also play a role in the structural transition from α-helices to α-sheets that may occur as nanofibers developed. Further understanding how to control this water structure could lead to novel ways of drug delivery through tuning the molecular mobility of water within the 3-D matrix itself.