A Competition of Secondary and Primary Nucleation Controls Amyloid Fibril Formation of the Parathyroid Hormone

Abstract

Functional amyloids belong to an increasing class of non‐toxic biologic material, in contrast to the prominent disease‐related amyloids. Herein, this work reports on the fibril formation of the parathyroid hormone PTH84 as a representative candidate following the same generic principles of primary and secondary nucleation. Employing Thioflavin T monitored kinetics analyses and negative‐staining transmission electron microscopy, an intricate, concentration dependent behavior of time dependent generation and morphologies of PTH84 fibrils are found. While at low peptide concentrations, fibril formation is driven by surface catalyzed secondary nucleation, an increased amount of peptides cause a negative feedback on fibril elongation and secondary nucleation. Moreover, the source of primary nuclei is found to regulate the overall macroscopic fibrillation. As a consequence, the concentration dependent competition of primary versus secondary nucleation pathways is found to dominate the mechanism of fibril generation. This work is able to hypothesize an underlying monomer‐oligomer equilibrium providing high‐order species for primary nucleation and, additionally, negatively affecting the available monomer pool.

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

“…In the high c 0 range, an additional state was observed with increased r H (PFG‐NMR and FCCS), higher r G (SAXS‐EOM) and larger r ee (smFRET) compared to the lower c 0 regimes. Within the framework of our previous reported mechanism of PTH 84 fibril formation, [23] we propose that the trimer or tetramer acts as precursor for primary nucleation at low c 0 (<310 μM, regime III), since these oligomers only appeared above c crit . The previously calculated reaction order for primary nucleation n 1 , being related to the physical nucleus size, of 2≤ n 1 ≤4 supports such oligomer sizes [23] .…”

“…Within the framework of our previous reported mechanism of PTH 84 fibril formation, [23] we propose that the trimer or tetramer acts as precursor for primary nucleation at low c 0 (<310 μM, regime III), since these oligomers only appeared above c crit . The previously calculated reaction order for primary nucleation n 1 , being related to the physical nucleus size, of 2≤ n 1 ≤4 supports such oligomer sizes [23] . Interestingly, fibrillation was found to proceed with lag‐times ( t lag ) of approximately 175 h ( c 0 =100 μM) decreasing with increasing c 0 until fibrillation apparently becomes c 0 independent at 250 μM ( t lag ≈110 h).…”

“…The high‐order oligomer at regime IV, as previously hypothesized from kinetic experiments and supported by smFRET, FCCS and PFG‐NMR in this work, is suspected to be the nucleation precursor at high c 0 (>310 μM). For this c 0 regime we found a switch of the reaction order of primary nucleation towards n 1 =18.5, enabling a competition with secondary nucleation [23] . This led to a considerable reduction of the lag‐time to about 60 h for c 0 =500 μM and further to ca.…”

“…The concentration of free peptide in the thermodynamic equilibrium with fibrils is also reflected by c crit [20] . This can be experimentally determined e. g. by sedimentation of the fibrils with a subsequent quantification of the soluble protein fraction or by diluting fibrils which, as a consequence, release monomers until c crit is reached [21–23] . Protein solutions exceeding c crit are supersaturated, metastable and kinetically soluble [24–25] .…”

“…The N‐terminal part of PTH 84 displays an α‐helical propensity while the C‐terminal part remains disordered [39–42] . PTH 84 offers a reversible fibrillation system with c crit in a μM concentration range which is easily accessible by a variety of biophysical techniques [21–23] . The secondary structure and oligomerization propensity of PTH 84 below, near and above c crit are investigated at conditions at which the peptide self‐assembles into fibrils to characterize potential transitions as a consequence of supersaturation.…”