Coupling proteins, with deadly consequences.

Phage protein E shuts down cell wall biosynthesis to kill bacteria.

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.

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.

Early Stage UV-B Induced Molecular Modifications of Human Eye Lens γD-Crystallin.

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, ß-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation.

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.

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.

Monitoring protein unfolding transitions by NMR-spectroscopy.

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.

Inactivation of Parathyroid Hormone: Perspectives of Drug Discovery to Combating Hyperparathyroidism.

Hormonal coordination is tightly regulated within the human body and thus regulates human physiology. The parathyroid hormone (PTH), a member of the endocrine system, regulates the calcium and phosphate level within the human body. Under non-physiological conditions, PTH levels get upregulated (hyperparathyroidism) or downregulated (hypoparathyroidism) due to external or internal factors. In case of hyperparathyroidism, elevated PTH stimulates cellular receptors present in the bones, kidneys, and intestines to increase the blood calcium level, leading to calcium deposition. This eventually causes various symptoms, including kidney stones. Currently, there is no known medication that directly targets PTH in order to suppress its function. Therefore, it is of great interest to find novel small molecules or any other means that can modulate PTH function. The molecular signaling of PTH starts by binding its N-terminus to the G-protein coupled PTH1/2 receptor. Therefore, any intervention that affects the N-terminus of PTH could be a lead candidate for treating hyperparathyroidism. As a proof-of-concept, there are various possibilities to inhibit molecular PTH function by (i) a small molecule, (ii) N-terminal PTH phosphorylation, (iii) fibril formation and (iv) residue-specific mutations. These modifications put PTH into an inactive state, which will be discussed in detail in this review article. We anticipate that exploring small molecules or other means that affect the N-terminus of PTH could be lead candidates in combating hyperparathyroidism.

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1.

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613-694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1613-694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.

Folding and Stability of Ankyrin Repeats Control Biological Protein Function.

Ankyrin repeat proteins are found in all three kingdoms of life. Fundamentally, these proteins are involved in protein-protein interaction in order to activate or suppress biological processes. The basic architecture of these proteins comprises repeating modules forming elongated structures. Due to the lack of long-range interactions, a graded stability among the repeats is the generic properties of this protein family determining both protein folding and biological function. Protein folding intermediates were frequently found to be key for the biological functions of repeat proteins. In this review, we discuss most recent findings addressing this close relation for ankyrin repeat proteins including DARPins, Notch receptor ankyrin repeat domain, IκBα inhibitor of NFκB, and CDK inhibitor p19INK4d. The role of local folding and unfolding and gradual stability of individual repeats will be discussed during protein folding, protein-protein interactions, and post-translational modifications. The conformational changes of these repeats function as molecular switches for biological regulation, a versatile property for modern drug discovery.

Lipid-Dependent Interaction of Human N-BAR Domain Proteins with Sarcolemma Mono- and Bilayers.

The N-BAR domain of the human Bin1 protein is indispensable for T-tubule biogenesis in skeletal muscles. It binds to lipid mono- and bilayers that mimic the sarcolemma membrane composition, and it transforms vesicles into uniform tubules by generating a decorating protein scaffold. We found that Δ(1-33)BAR, lacking the N-terminal amphipathic helix (H0), and H0 alone bind to sarcolemma monolayers, although both proteins are not able to tubulate sarcolemma vesicles. By variation of the lipid composition, we elucidated the role of PI(4,5)P2, cholesterol, and an asymmetric sarcolemma composition for Bin1-N-BAR binding and sarcolemma tubulation. Our results indicate that Bin1-N-BAR binding is low in the absence of PI(4,5)P2 and it is affected by additional changes in the negative headgroup charge and lipid acyl chain composition. However, it is not dependent on the cholesterol content. The results from Langmuir monolayer experiments are complementary to lipid bilayer studies using electron microscopy that provides information on membrane curvature generation.

Insights into the secondary structures of lactam N-substituted stapled peptides.

Stapled peptides derived from the Ugi macrocyclization comprise a special class of cyclopeptides with an N-substituted lactam bridge cross-linking two amino acid side chains. Herein we report a comprehensive analysis of the structural factors influencing the secondary structure of these cyclic peptides in solution. Novel insights into the s-cis/s-trans isomerism and the effect of N-functionalization on the conformation are revealed.

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.

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.

In-Cell NMR: Analysis of Protein-Small Molecule Interactions, Metabolic Processes, and Protein Phosphorylation.

Nuclear magnetic resonance (NMR) spectroscopy enables the non-invasive observation of biochemical processes, in living cells, at comparably high spectral and temporal resolution. Preferably, means of increasing the detection limit of this powerful analytical method need to be applied when observing cellular processes under physiological conditions, due to the low sensitivity inherent to the technique. In this review, a brief introduction to in-cell NMR, protein-small molecule interactions, posttranslational phosphorylation, and hyperpolarization NMR methods, used for the study of metabolites in cellulo, are presented. Recent examples of method development in all three fields are conceptually highlighted, and an outlook into future perspectives of this emerging area of NMR research is given.

Hyperbolic Pressure-Temperature Phase Diagram of the Zinc-Finger Protein apoKti11 Detected by NMR Spectroscopy.

For a comprehensive understanding of the thermodynamic state functions describing the stability of a protein, the influence of the intensive properties of temperature and pressure has to be known. With the zinc-finger-containing Kti11, we found a suitable protein for this purpose because folding and unfolding transitions occur at an experimentally accessible temperature (280-330 °K) and pressure (0.1-240 MPa) range. We solved the crystal structure of the apo form of Kti11 to reveal two disulfide bonds at the metal-binding site, which seals off a cavity in the ß-barrel part of the protein. From a generally applicable proton NMR approach, we could determine the populations of folded and unfolded chains under all conditions, leading to a hyperbolic pressure-temperature phase diagram rarely observed for proteins. A global fit of a two-state model to all derived populations disclosed reliable values for the change in Gibbs free energy, volume, entropy, heat capacity, compressibility, and thermal expansion upon unfolding. The unfolded state of apoKti11 has a lower compressibility compared to the native state and a smaller volume at ambient pressure. Therefore, a pressure increase up to 200 MPa reduces the population of the native state, and above this value, the native population increases again. Pressure-induced chemical-shift changes in two-dimensional 1H-15N NMR spectra could be employed for a molecular interpretation of the thermodynamic properties of apoKti11.

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.

A Multicomponent Stapling Approach to Exocyclic Functionalized Helical Peptides: Adding Lipids, Sugars, PEGs, Labels, and Handles to the Lactam Bridge.

Peptide stapling is traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization chemistries. In an endeavor to add a diversity-generating tool to this repertoire, we introduce a multicomponent stapling approach enabling the simultaneous stabilization of helical secondary structures and the exocyclic N-functionalization of the side chain-tethering lactam bridge. This is accomplished by means of a novel solid-phase methodology comprising, for the first time, the on-resin Ugi reaction-based macrocyclization of peptide side chains bearing amino and carboxylic acid groups. The exocyclic diversity elements arise from the isocyanide component used in the Ugi multicomponent stapling protocol, which allows for the incorporation of relevant fragments such as lipids, sugars, polyethylene glycol, fluorescent labels, and reactive handles. We prove the utility of such exocyclic reactive groups in the bioconjugation of a maleimide-armed lactam-bridged peptide to a carrier protein. The on-resin multicomponent stapling proved efficient for the installation of not only one, but also two consecutive lactam bridges having either identical or dissimilar N-functionalities. The easy access to helical peptides with a diverse set of exocyclic functionalities shows prospect for applications in peptide drug discovery and chemical biology.