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.

Lighting up Nobel Prize-winning studies with protein intrinsic disorder.

Intrinsically disordered proteins and regions (IDPs and IDRs) and their importance in biology are becoming increasingly recognized in biology, biochemistry, molecular biology and chemistry textbooks, as well as in current protein science and structural biology curricula. We argue that the sequence → dynamic conformational ensemble → function principle is of equal importance as the classical sequence → structure → function paradigm. To highlight this point, we describe the IDPs and/or IDRs behind the discoveries associated with 17 Nobel Prizes, 11 in Physiology or Medicine and 6 in Chemistry. The Nobel Laureates themselves did not always mention that the proteins underlying the phenomena investigated in their award-winning studies are in fact IDPs or contain IDRs. In several cases, IDP- or IDR-based molecular functions have been elucidated, while in other instances, it is recognized that the respective protein(s) contain IDRs, but the specific IDR-based molecular functions have yet to be determined. To highlight the importance of IDPs and IDRs as general principle in biology, we present here illustrative examples of IDPs/IDRs in Nobel Prize-winning mechanisms and processes.

Inherent Adaptivity of Alzheimer Peptides to Crowded Environments.

Amyloid ß (Aß) is the major constituent in senile plaques of Alzheimer's disease in which peptides initially undergo structural conversions to form elongated fibrils. The impact of crowding on the fibrillation pathways of Aß40 and Aß42 , the most common peptide isoforms are studied. PEG and Ficoll are used as model crowders to mimic a macromolecular enriched surrounding. The fibrillar growth is monitored with the help of ThT-fluorescence assays in order to extract two rates describing primary and secondary processes of nucleation and growth. Techniques as fluorescence correlation spectroscopy and analytical ultracentrifugation are used to discuss oligomeric states; fibril morphologies are investigated using negative-staining transmission electron microscopy. While excluded volume effects imposed by macromolecular crowding are expected to always increase rates of intermolecular interactions and structural conversion, a vast variety of effects are found depending on the peptide, the crowder, or ionic strength of the solution. While investigations of the obtained rates with respect to a reactant-occluded model are capable to display specific surface interactions with the crowder, the employment of crystallization-like models reveal the crowder-induced entropic gain with Δ Δ G fib crow = - 116 ± 21 k $\Delta \Delta G_{\text{fib}}^{\text{crow}}=-116\pm 21\; k$ J mol-1 per volume fraction of the crowder.

Heparin promotes rapid fibrillation of the basic parathyroid hormone at physiological pH.

In acidic secretory granules of mammalian cells, peptide hormones including the parathyroid hormone are presumably stored in the form of functional amyloid fibrils. Mature PTH, however, is considerably positively charged in acidic environments, a condition known to impede unassisted self-aggregation into fibrils. Here, we studied the role of the polyanion heparin on promoting fibril formation of PTH. Employing ITC, CD spectroscopy, NMR, SAXS, and fluorescence-based assays, we could demonstrate that heparin binds PTH with submicromolar affinity and facilitates its conversion into fibrillar seeds, enabling rapid formation of amyloid fibrils under acidic conditions. In the absence of heparin, PTH remained in a soluble monomeric state. We suspect that heparin-like surfaces are required in vivo to convert PTH efficiently into fibrillar deposits.