Investigating the Self-Aggregation of a Polyalanine Peptide: Kinetics and Isotope Edited Studies

Abstract

Exploring the extended β-sheet structures associated with protein aggregation is pivotal for a clear understanding of the factors governing fibril formation. We have recently identified three short alanine based peptides with unusual capabilities to form amyloid-like fibrils in solution, namely Ac-AAAAKAAY-NH2 (AKY8) and (AAKA)4 and (AARA)4. Oligoalanine peptides of these sizes typically exhibit either a statistical coil structure in aqueous solution, or if long enough, α-helical conformations. The fibril formation of AKY8 can be monitored by a strongly enhanced amide I’ Vibrational Circular Dichroism (VCD) signal, which we have shown is due to helical twisting of stacked β-sheets. Due to the inability of the non-tyrosine variant of AKY8, (i.e. AAAAKAA) to aggregate, we hypothesize that the self-aggregation of AKY8 is promoted by cation-π interaction between lysine and tyrosine side-chains. In order to further examine this aggregation mechanism we use Isotope-Edited Infrared (IR) and Vibrational Circular Dichroism Spectroscopy (VCD), which provide site-specific structural information and intra-stand registry of the peptide. First results indicate that there is indeed an in-register arrangement of parallel β-strands in AKY8 with lysine and tyrosine residues aligned between stacks. We also investigate the surprising aggregation of the amphiphilic and positively charged (AAKA)4 peptide which aggregates and forms hydrogels at millimolar concentrations and acidic pH. Interestingly, kinetic studies on this peptide indicate that β-sheet aggregation lacks a nucleation phase. At low concentrations, these peptides decay into disordered structures, according to ECD and IR spectra. At higher peptide concentrations however, this decay is not observed, while some relaxation is still operative, possibly due to a rearrangement of the sheet-like structure. We find that the arginine variant of this peptide, i.e. (AARA)4, also aggregates to form hydrogels, however, this peptide has a long lag phase.