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.

Synthesis and Aggregation of Polymer-Amyloid ß Conjugates.

Modulating the assembly of medically relevant peptides and proteins via macromolecular engineering is an important step in modifying their overall pathological effects. The synthesis of polymer-peptide conjugates composed of the amyloidogenic Alzheimer peptide, Aß1-40 , and poly(oligo(ethylene glycol)m acrylates) (m = 2,3) with different molecular weights (Mn = 1400-6600 g mol-1 ) is presented here. The challenging conjugation of a synthetic polymer to an in situ aggregating protein is established via two different coupling strategies, only successful for polymers with molecular weights not exceeding 6600 g mol-1 , relying on resin-based synthesis or solution-based coupling chemistries. The conjugates are characterized by high-performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The aggregation of these polymer-Aß1-40 conjugates, as monitored via thioflavine-T (ThT)-fluorescence spectroscopy, is accelerated mainly upon attaching the polymers. However, the appearance of the observed fibrils is different from those composed of native Aß1-40, specifically with respect to length and morphology of the obtained aggregates. Instead of long, unbranched fibrils characteristic for Aß1-40 , bundles of short aggregates are observed for the conjugates. Finally, the ThT kinetics and morphologies of Aß1-40 fibrils formed in the presence of the conjugates give some mechanistic insights.

ß-Turn mimetic synthetic peptides as amyloid-ß aggregation inhibitors.

Aggregation of amyloid peptides results in severe neurodegenerative diseases. While the fibril structures of Aß40 and Aß42 have been described recently, resolution of the aggregation pathway and evaluation of potent inhibitors still remains elusive, in particular in view of the hairpin-region of Aß40. We here report the preparation of beta-turn mimetic conjugates containing synthetic turn mimetic structures in the turn region of Aß40 and Aß16-35, replacing 2 amino acids in the turn-region G25 - K28. The structure of the turn mimic induces both, acceleration of fibrillation and the complete inhibition of fibrillation, confirming the importance of the turn region on the aggregation. Replacing position G25-S26 provided the best inhibition effect for both beta-turn mimetics, the bicyclic BTD 1 and the aromatic TAA 2, while positions N27-K28 and V24-G25 showed only weaker or no inhibitory effects. When comparing different turn mimetics at the same position (G25-S26), conjugate 1a bearing the BTD turn showed the best inhibition of Aß40 aggregation, while 5-amino-valeric acid 4a showed the weakest effect. Thus there is a pronounced impact on fibrillation with the chemical nature of the embedded beta-turn-mimic: the conformationally constrained turns 1 and 2 lead to a significantly reduced fibrillation, even inhibiting fibrillation of native Aß40 when added in amounts down to 1/10, whereas the more flexible beta-turn-mimics 4-amino-benzoic acid 3a and 5-amino-valeric acid 4a lead to enhanced fibrillation. Toxicity-testing of the most successful conjugate showed only minor toxicity in cell-viability assays using the N2a cell line. Structural downsizing lead to the short fragment BTD/peptide Aß16-35 as inhibitor of the aggregation of Aß40, opening large potential for further small peptide based inhibitors.

How Fluorescent Tags Modify Oligomer Size Distributions of the Alzheimer Peptide.

Within the complex aggregation process of amyloidogenic peptides into fibrils, early stages of aggregation play a central role and reveal fundamental properties of the underlying mechanism of aggregation. In particular, low-molecular-weight aggregates of the Alzheimer amyloid-ß peptide (Aß) have attracted increasing interest because of their role in cytotoxicity and neuronal apoptosis, typical of aggregation-related diseases. One of the main techniques used to characterize oligomeric stages is fluorescence spectroscopy. To this end, Aß peptide chains are functionalized with fluorescent tags, often covalently bound to the disordered N-terminus region of the peptide, with the assumption that functionalization and presence of the fluorophore will not modify the process of self-assembly nor the final fibrillar structure. In this investigation, we systematically study the effects of four of the most commonly used fluorophores on the aggregation of Aß (1-40). Time-resolved and single-molecule fluorescence spectroscopy have been chosen to monitor the oligomer populations at different fibrillation times, and transmission electron microscopy, atomic force microscopy and x-ray diffraction to investigate the structure of mature fibrils. Although the structures of the fibrils were only slightly affected by the fluorescent tags, the sizes of the detected oligomeric species varied significantly depending on the chosen fluorophore. In particular, we relate the presence of high-molecular-weight oligomers of Aß (1-40) (as found for the fluorophores HiLyte 647 and Atto 655) to net-attractive, hydrophobic fluorophore-peptide interactions, which are weak in the case of HiLyte 488 and Atto 488. The latter leads for Aß (1-40) to low-molecular-weight oligomers only, which is in contrast to Aß (1-42). The disease-relevant peptide Aß (1-42) displays high-molecular-weight oligomers even in the absence of significant attractive fluorophore-peptide interactions. Hence, our findings reveal the potentially high impact of the properties of fluorophores on transient aggregates, which needs to be included in the interpretation of experimental data of oligomers of fluorescently labeled peptides.

Probing Polymer Chain Conformation and Fibril Formation of Peptide Conjugates.

Covalent conjugates between a synthetic polymer and a peptide hormone were used to probe the molecular extension of these macromolecules and how the polymer modifies the fibril formation of the hormone. NMR spectroscopy of 15 N labeled parathyroid hormone (PTH) was employed to visualize the conformation of the conjugated synthetic polymer, triggered by small temperature changes via its lower critical solution temperature. A shroud-like polymer conformation dominated the molecular architecture of the conjugated chimeras. PTH readily forms amyloid fibrils, which is probably the physiological storage form of the hormone. The polyacrylate based polymers stimulated the nucleation processes of the peptide.

Modulation of amyloid ß peptide aggregation by hydrophilic polymers.

A substantial number of diseases leading to loss of neurologic functions such as Morbus Alzheimer, Morbus Parkinson, or Chorea Huntington are related to the fibrillation of particular amyloidogenic peptides. In vitro amyloid fibrillation strongly depends on admixture with other proteins and peptides, lipids, nanoparticles, surfactants and polymers. We investigated amyloid-beta 1-40 peptide (Aß1-40) fibrillation in mixture with thermoresponsive poly(oligo(ethylene glycol)macrylates), in which the polymer's hydrophobicity is tuned by variation of the number of ethylene glycol-units in the side chain (m = 1-9), the end groups (B = butoxy; C = carboxy; D = dodecyl; P = pyridyldisulfide) and the degree of polymerization (n) of the polymers. The polymers were prepared via RAFT-polymerization, obtaining a broad range of molecular masses (Mn = 700 to 14 600 g mol-1 kDa-1, polydispersity indices PDI = 1.10 to 1.25) and tunable cloud point temperatures (Tcp), ranging from 42.4 °C to 80 °C, respectively. Proper combination of hydrophobic end groups with hydrophilic side chains of the polymer allowed to alter the hydrophilicity/hydrophobicity of these polymers, which is shown to enhance Aß1-40 aggregation significantly in case of the endgroup D (with n = 16, 23, 56). We observed that the less hydrophilic polymers (m = 1-2) were able to both decrease and elongate the lag (tlag) and characteristic times (tchar) of Aß1-40 fibril formation in dependence of their end groups, molecular mass and hydrophilicity. On the other hand, highly hydrophilic polymers (m = 3, 5, 9) either decreased, or only marginally influenced the lag and characteristic times of Aß1-40 fibrillation, in all cases forming ß-sheet rich fibrils as observed by TEM and CD-spectroscopy. Our results support that balanced hydrophobic and hydrophilic interactions of a polymer with Aß1-40 is important for inhibiting amyloid-formation pathways.

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid ß (1-40).

A small library of rationally designed amyloid ß [Aß(1-40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aß(1-40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid-state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aß(1-40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aß fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross-ß structure.

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

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 pro-sequence of parathyroid hormone prevents premature amyloid fibril formation.

The parathyroid hormone (PTH) regulates the calcium and phosphate level in blood after secretion from parathyroid chief cells. The pre- and pro-sequences of precursor preproPTH get cleaved during PTH maturation. In secretory granules, PTH forms functional amyloids. Using thioflavin T fibrillation assays, circular dichroism, NMR spectroscopy, and cellular cAMP activation, we show that the pro-sequence prevents premature fibrillation by impairing primary nucleation because of Coulomb repulsion of positively charged residues. Under seeding or high salt conditions or in the presence of heparin at pH 5.5, proPTH fibril formation is delayed, but the monomer release properties are conserved. ProPTH can still activate in cellulo PTH receptor 1 but with impaired potency. These findings give some perspectives on medical applications of PTH in hormone therapy.

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.

Modulating the Fibrillization of Parathyroid-Hormone (PTH) Peptides: Azo-Switches as Reversible and Catalytic Entities.

We here report a novel strategy to control the bioavailability of the fibrillizing parathyroid hormone (PTH)-derived peptides, where the concentration of the bioactive form is controlled by an reversible, photoswitchable peptide. PTH1-84, a human hormone secreted by the parathyroid glands, is important for the maintenance of extracellular fluid calcium and phosphorus homeostasis. Controlling fibrillization of PTH1-84 represents an important approach for in vivo applications, in view of the pharmaceutical applications for this protein. We embed the azobenzene derivate 3-{[(4-aminomethyl)phenyl]diazenyl}benzoic acid (3,4'-AMPB) into the PTH-derived peptide PTH25-37 to generate the artificial peptide AzoPTH25-37 via solid-phase synthesis. AzoPTH25-37 shows excellent photostability (more than 20 h in the dark) and can be reversibly photoswitched between its cis/trans forms. As investigated by ThT-monitored fibrillization assays, the trans-form of AzoPTH25-37 fibrillizes similar to PTH25-37, while the cis-form of AzoPTH25-37 generates only amorphous aggregates. Additionally, cis-AzoPTH25-37 catalytically inhibits the fibrillization of PTH25-37 in ratios of up to one-fifth. The approach reported here is designed to control the concentration of PTH-peptides, where the bioactive form can be catalytically controlled by an added photoswitchable peptide.

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.