Voigt Bruno, Bhatia Twinkle, Hesselbarth Julia, Baumann Monika, Schmidt Carla, Ott Maria, Balbach Jochen
Martin Luther University Halle-Wittenberg, Institute of Physics, Betty-Heimann-Straße 7, 06120, Halle, Germany.
Martin Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Straße 3, 06120, Halle, Germany.
Chemphyschem. 2023 Oct 4;24(19):e202300439. doi: 10.1002/cphc.202300439. Epub 2023 Aug 3.
Nucleation and growth of amyloid fibrils were found to only occur in supersaturated solutions above a critical concentration (c ). The biophysical meaning of c remained mostly obscure, since typical low values of c in the sub-μM range hamper investigations of potential oligomeric states and their structure. Here, we investigate the parathyroid hormone PTH as an example of a functional amyloid fibril forming peptide with a comparably high c 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 c as possible precursors for primary fibril nucleation. Furthermore, the soluble state found in equilibrium with fibrils adopts to the prenucleation state present at c . 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 c of amyloid fibril formation of PTH 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 c of PTH 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.
淀粉样纤维的成核和生长仅在高于临界浓度（c）的过饱和溶液中发生。c的生物物理意义大多仍不清楚，因为在亚微摩尔范围内典型的低c值妨碍了对潜在寡聚体状态及其结构的研究。在此，我们以甲状旁腺激素PTH为例进行研究，它是一种功能性淀粉样纤维形成肽，其c相对较高，为67±21μM。我们描述了一个复杂的、浓度依赖性的不同大小和二级结构组成的寡聚体预成核聚集体，并强调在c时三聚体和四聚体的出现可能是原纤维成核的前体。此外，与纤维处于平衡状态的可溶状态与c时存在的预成核状态相适应。我们的研究揭示了与临界浓度直接相关的淀粉样形成早期事件，并强调寡聚体形成是纤维成核的关键特征。我们的结果有助于更深入地理解过饱和肽溶液的决定因素。在当前研究中，我们提出了一种生物物理方法，从二级结构、簇大小和寡聚体形成过程中残基分辨的分子间相互作用方面研究PTH淀粉样纤维形成的c。在整个研究的浓度范围（1μM至500μM）内，我们发现了不同的寡聚化状态，它们对原纤维成核的贡献能力不同，且具有浓度依赖性平衡。在此背景下，我们确定了先前描述的PTH的c标志着同三聚体/四聚体形成的最低浓度。这些研究使我们能够表征各种寡聚体状态的分子相互作用，这些相互作用在功能性淀粉样形成肽的延迟期进一步转化为能够延伸的纤维核。
