Mechanisms of early amyloid formation and the confining influence of macromolecular crowding

The structural flexibility of amyloid peptides comprises the extraordinary property to self-assemble into extended fibrillar structures. Molecular insights into the structure and composition of prefibrillar aggregates are essential for the understanding of the overall aggregation process. This project aims to get a more detailed understanding on the early prefibrillar growth process and the influence of the contiguous environment by the use of single-molecule studies. Resulting distributions of small transient aggregates (named oligomers) along the time course of the fibrillation can be used to study the pathways of aggregation. Specific properties of the detected oligomers, e.g. the propensity to disassemble or to interact with hydrophobic surfaces can be used to study their thermodynamic stabilities and increased hydrophobicity. Moreover, we could demonstrate that the photophysical properties of the fluorescence tags itself, e.g. the fluorescence lifetime, can be used to distinguish between (potentially unstructured) oligomers and fibrillar assemblies. In addition, aggregates which are not consumed by the process of fibrillation are indicative for off-pathway processes and a reduced efficiency of fibrillation.
In the previous funding period we established methods of single-molecule and time-resolved fluorescence spectroscopy to study the early stages of amyloid self-assembly and investigated the influence of commonly used fluorescence tags on the properties of oligomers and fibrils of N-terminal labeled A(1-40) and A(1-42)-peptides, which are related to the Alzheimer disease. The solubility of the formed oligomers as well as their size distributions were found to be not only influenced by the length of the peptides but also by specific fluorophore-peptide interactions. Most importantly, we found a fluorescence tag with the least impact on the aggregation of the peptide, which will be used in future work. In preparation for detailed investigations of the effects of macromolecular crowding on the early
aggregates and the overall process of fibrillation, we studied the properties of highly concentrated solutions of globular proteins by a combined approach of small-angle X-ray scattering experiments, NMR and fluorescence correlation spectroscopy.
In the next funding period we want to extend our study into two directions: Firstly, we plan to apply our methodological expertise to a new peptide, the human parathyroid hormone (PTH) which belongs to the family of functional amyloids, and, secondly, we want to study the effects of external constraints on amyloid aggregation. In contrast to the A-peptides, the aggregation process of PTH is reversible, which will enable us to learn more about one potential key for the non-toxicity of the aggregates - the lower thermodynamic stability. In particular we want to determine the size of the critical nucleus and study its relation to monomer concentration, aggregate size and the overall propensity for disassembly. An indication for reduced stabilities of PTH oligomers are the comparably high monomer concentrations which are required for fibrillation. Complementary to fluorescence spectroscopy we will continue to use small and wide-angle X-ray scattering (SAXS,WAXS) and imaging techniques (Transmission electron microscopy (TEM), atomic force microscopy (AFM)) to characterize size, structure and morphology of the formed aggregates. In the second part of the project we will address the influence of external constraints and (un)specific interactions by using different proteins and crowding agents. Here, binding and unbinding kinetics of the amyloid protein and its oligomers with proteins crowders will be investigated using single-molecule Förster-resonance energy transfer (FRET) methods in solution. These experiments will help to quantify specific interactions and excluded volume effects. The comparison of the aggregation mechanisms of the A-peptides and PTH, as well as their potentially different response to external constraints, will finally deepen the general understanding of the complex interplay of amyloid aggregates with their close environment.