The Prenucleation Equilibrium of the Parathyroid Hormone Determines the Critical Aggregation Concentration and Amyloid Fibril Nucleation.

Nucleation and growth of amyloid fibrils were found to only occur in supersaturated solutions above a critical concentration (ccrit ). The biophysical meaning of ccrit remained mostly obscure, since typical low values of ccrit in the sub-µM range hamper investigations of potential oligomeric states and their structure. Here, we investigate the parathyroid hormone PTH84 as an example of a functional amyloid fibril forming peptide with a comparably high ccrit of 67±21 µM. We describe a complex concentration dependent prenucleation ensemble of oligomers of different sizes and secondary structure compositions and highlight the occurrence of a trimer and tetramer at ccrit as possible precursors for primary fibril nucleation. Furthermore, the soluble state found in equilibrium with fibrils adopts to the prenucleation state present at ccrit . Our study sheds light onto early events of amyloid formation directly related to the critical concentration and underlines oligomer formation as a key feature of fibril nucleation. Our results contribute to a deeper understanding of the determinants of supersaturated peptide solutions. In the current study we present a biophysical approach to investigate ccrit of amyloid fibril formation of PTH84 in terms of secondary structure, cluster size and residue resolved intermolecular interactions during oligomer formation. Throughout the investigated range of concentrations (1 µM to 500 µM) we found different states of oligomerization with varying ability to contribute to primary fibril nucleation and with a concentration dependent equilibrium. In this context, we identified the previously described ccrit of PTH84 to mark a minimum concentration for the formation of homo-trimers/tetramers. These investigations allowed us to characterize molecular interactions of various oligomeric states that are further converted into elongation competent fibril nuclei during the lag phase of a functional amyloid forming peptide.