Developing nonperturbative spectroscopic approaches for characterizing heterogenous ensembles during protein self-assembly

Abstract

Peptide self-assembly has recently been explored to create novel biomaterials with direct applications to medicine. The complex relationship of intra- and intermolecular forces that drive self-assembly lead to an wide array of aggregated structures that can be difficult to predict based solely on the primary sequence.The ability to detect and characterize multiple secondary structures or polymorphs within peptide and protein aggregates is crucial to the rational design of novel biomaterials, treatment and prevention of amyloidogenic diseases, and many other applications. Here, I investigate the effects of N-terminal acetylation on model amphiphilic peptides, KFE8 and AcKFE8, using two-dimensional infrared spectroscopy and site-specific isotope labeling. Two-dimensional infrared spectra reveal that AcKFE8 aggregates comprise two distinct β-sheet structures while KFE8 aggregates comprise only one of these structures. By calculating the actual transition dipole strengths from two-dimensional infrared spectra, we are able to detect additional distinct β-sheet configurations at early time-points during aggregation. This yields a label-free method of polymorph detection within seemingly homogenous protein aggregates. These techniques are applied to wild-type and acetylated variants of amyloid beta 1-42, the protein associated with Alzheimer’s disease, in order to characterize the secondary structure composition and aggregation kinetics of more complex and cytotoxic amyloidogenic proteins during self-assembly. The results presented herein highlight the importance of understanding the residue-level structural variations that result from subtle changes in peptide sequence and demonstrate a powerful spectroscopic method to distinguish multiple oligomeric and polymorphic motifs during proteinself-assembly.