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.

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.

Phosphorylation-induced unfolding regulates p19INK4d during the human cell cycle.

Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs). The ankyrin-repeat protein p19INK4d functions as a key regulator of G1/S transition; however, its molecular mode of action is unknown. Here, we combine cell and structural biology methods to unravel the mechanism by which p19INK4d controls cell cycle progression. We delineate how the stepwise phosphorylation of p19INK4d Ser66 and Ser76 by cell cycle-independent (p38) and -dependent protein kinases (CDK1), respectively, leads to local unfolding of the three N-terminal ankyrin repeats of p19INK4d This dissociates the CDK6-p19INK4d inhibitory complex and, thereby, activates CDK6. CDK6 triggers entry into S-phase, whereas p19INK4d is ubiquitinated and degraded. Our findings reveal how signaling-dependent p19INK4d unfolding contributes to the irreversibility of G1/S transition.

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.

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.

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.

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.

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.

Quantitative NMR study of heat-induced aggregation of eye-lens crystallin proteins under crowding conditions.

The eye lens contains a highly concentrated, polydisperse mixture of crystallins, and a loss in transparency during cataract formation is attributed to the aggregation of these proteins. Most biochemical and biophysical studies of crystallins have been performed in diluted samples because of various physical limitations of the respective method at physiological concentrations of up to 200-400 mg/mL. We introduce a straightforward proton NMR transverse relaxometry method to quantify simultaneously proteins in the dissolved and aggregated states at these elevated concentrations, because these states significantly differ in their transverse relaxation properties. The key feature of this method is a direct observation of the protein signal in a wide range of relaxation delays, from few microseconds up to few hundred milliseconds. We applied this method to follow heat-induced aggregation of bovine α- and γB-crystallin between 60 and 200 mg/mL. We find that at 60 °C, a temperature where both crystallins still comprise a native tertiary structure, γB-crystallin aggregated at these high protein concentrations with a time constant of about 30-40 h. α-crystallin remained soluble at 60 mg/mL but formed a transparent gel at 200 mg/mL. This quantitative NMR method can be applied to investigations of other proteins and their mixtures under various aggregation conditions.

Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response.

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of auxin/indole-3-acetic acid (AUX/IAA)-inducible repressors, which directly bind to transcriptional auxin response factors (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like ß-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼ 6.4 µM) were determined by isothermal titration calorimetry. In silico protein-protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein-protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.

Structure-Based Insights into the Dynamics and Function of Two-Domain SlpA from Escherichia coli.

SlpA (SlyD-like protein A) comprises two domains, a FK506 binding domain (FKBP fold) of moderate prolyl cis/trans-isomerase activity and an inserted in flap (IF) domain that hosts its chaperone activity. Here we present the nuclear magnetic resonance (NMR) solution structure of apo Escherichia coli SlpA determined by NMR that mirrors the structural properties seen for various SlyD homologues. Crucial structural differences in side-chain orientation arise for F37, which points directly into the hydrophobic core of the active site. It forms a prominent aromatic stacking with F15, one of the key residues for PPIase activity, thus giving a possible explanation for the inherently low PPIase activity of SlpA. The IF domain reveals the highest stability within the FKBP-IF protein family, most likely arising from an aromatic cluster formed by four phenylalanine residues. Both the thermodynamic stability and the PPIase and chaperone activity let us speculate that SlpA is a backup system for homologous bacterial systems under unfavorable conditions.

Inhibition of Aß(1-40) fibril formation by cyclophilins.

Cyclophilins interact directly with the Alzheimer's disease peptide Aß (amyloid ß-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aß binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aß(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aß(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aß(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aß(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aß fibril formation by cyclophilins.

Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane.

The 30kDa N-BAR domain of the human Bin1 protein is essential for the generation of skeletal muscle T-tubules. By electron cryo-microscopy and electron cryo-tomography with a direct electron detector, we found that Bin1-N-BAR domains assemble into scaffolds of low long-range order that form flexible membrane tubules. The diameter of the tubules closely matches the curved shape of the N-BAR domain, which depends on the composition of the target membrane. These insights are fundamental to our understanding of T-tubule formation and function in human skeletal muscle.

Real-time protein NMR spectroscopy and investigation of assisted protein folding.

BACKGROUND: During protein-folding reactions toward the native structure, short-lived intermediate states can be populated. Such intermediates expose hydrophobic patches and can self-associate leading to non-productive protein misfolding. A major focus of current research is the characterization of short-lived intermediates and how molecular chaperones enable productive folding. Real-time NMR spectroscopy, together with the development of advanced methods, is reviewed here and the potential these methods have to characterize intermediate states as well as interactions with molecular chaperone proteins at single-residue resolution is highlighted. SCOPE OF REVIEW: Various chaperone interactions can guide the protein-folding reaction and thus are important for protein structure formation, stability, and activity of their substrates. Chaperone-assisted protein folding, characterization of intermediates, and their molecular interactions using real-time NMR spectroscopy will be discussed. Additionally, recent advances in NMR methods employed for characterization of high-energy intermediates will be discussed. MAJOR CONCLUSIONS: Real-time NMR combines high resolution with kinetic information of protein reactions, which can be employed not only for protein-folding studies and the characterization of folding intermediates but also to investigate the molecular mechanisms of assisted protein folding. GENERAL SIGNIFICANCE: Real-time NMR spectroscopy remains an effective tool to reveal structural details about the interaction between chaperones and transient intermediates. Methodologically, it provides in-depth understanding of how kinetic intermediates and their thermodynamics contribute to the protein-folding reaction. This review summarizes the most recent advances in this field. This article is part of a Special Issue titled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Structural characterization of amyloid fibrils from the human parathyroid hormone.

Amyloid deposits are common in various tissues as a consequence of misfolded proteins. However, secretory protein and peptides are often stored in membrane coated granules as functional amyloids. In this article, we present a detailed characterization of in vitro generated amyloid fibrils from human parathyroid hormone (hPTH(1-84)). Fully mature fibrils could be obtained after a short lag phase within less than one hour at 65°C. These fibrils showed all characteristic of a cross-ß structure. Protease cleavage combined with mass spectrometry identified the central region of the peptide hormone involved in the fibril core formation. EGCG, an inhibitor of amyloid fibril formation, showed binding to residues in the peptide monomers corresponding to the later fibril core and thus explaining the inhibition of the fibril growth. Conformational and dynamic studies by solid-state NMR further corroborated the cross-ß core of the fibrils, but also identified highly mobile segments with a random coil structure not belonging to the rigid fibril core.

Coupling and Decoupling of Rotational and Translational Diffusion of Proteins under Crowding Conditions.

Molecular motion of biopolymers in vivo is known to be strongly influenced by the high concentration of organic matter inside cells, usually referred to as crowding conditions. To elucidate the effect of intermolecular interactions on Brownian motion of proteins, we performed (1)H pulsed-field gradient NMR and fluorescence correlation spectroscopy (FCS) experiments combined with small-angle X-ray scattering (SAXS) and viscosity measurements for three proteins, αB-crystalline (αBc), bovine serum albumin, and hen egg-white lysozyme (HEWL) in aqueous solution. Our results demonstrate that long-time translational diffusion quantitatively follows the expected increase of macro-viscosity upon increasing the protein concentration in all cases, while rotational diffusion as assessed by polarized FCS and previous multi-frequency (1)H NMR relaxometry experiments reveals protein-specific behavior spanning the full range between the limiting cases of full decoupling from (αBc) and full coupling to (HEWL) the macro-viscosity. SAXS was used to study the interactions between the proteins in solution, whereby it is shown that the three cases cover the range between a weakly interacting hard-sphere system (αBc) and screened Coulomb repulsion combined with short-range attraction (HEWL). Our results, as well as insights from the recent literature, suggest that the unusual rotational-translational coupling may be due to anisotropic interactions originating from hydrodynamic shape effects combined with high charge and possibly a patchy charge distribution.

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.