The three-dimensional structure of human ß-endorphin amyloid fibrils.

In the pituitary gland, hormones are stored in a functional amyloid state within acidic secretory granules before they are released into the blood. To gain a detailed understanding of the structure-function relationship of amyloids in hormone secretion, the three-dimensional (3D) structure of the amyloid fibril of the human hormone ß-endorphin was determined by solid-state NMR. We find that ß-endorphin fibrils are in a ß-solenoid conformation with a protonated glutamate residue in their fibrillar core. During exocytosis of the hormone amyloid the pH increases from acidic in the secretory granule to neutral level in the blood, thus it is suggested-and supported with mutagenesis data-that the pH change in the cellular milieu acts through the deprotonation of glutamate 8 to release the hormone from the amyloid. For amyloid disassembly in the blood, it is proposed that the pH change acts together with a buffer composition change and hormone dilution. In the pituitary gland, peptide hormones can be stored as amyloid fibrils within acidic secretory granules before release into the blood stream. Here, we use solid-state NMR to determine the 3D structure of the amyloid fiber formed by the human hormone ß-endorphin. We find that ß-endorphin fibrils are in a ß-solenoid conformation that is generally reminiscent of other functional amyloids. In the ß-endorphin amyloid, every layer of the ß-solenoid is composed of a single peptide and protonated Glu8 is located in the fibrillar core. The secretory granule has an acidic pH but, on exocytosis, the ß-endorphin fibril would encounter neutral pH conditions (pH 7.4) in the blood; this pH change would result in deprotonation of Glu8 to release the hormone peptide from the amyloid. Analyses of ß-endorphin variants carrying mutations in Glu8 support the role of the protonation state of this residue in fibril disassembly, among other environmental changes.

Line-Broadening in Low-Temperature Solid-State NMR Spectra of Fibrils.

The temperature-dependent resonance-line broadening of HET-s(218-289) in its amyloid form is investigated in the range between 110 K and 280 K. Significant differences are observed between residues in the structured hydrophobic triangular core, which are broadened the least and can be detected down to 100 K, and in the solvent-exposed parts, which are broadened the most and often disappear from the observed spectrum around 200 K. Below the freezing of the bulk water, around 273 K, the protein fibrils are still surrounded by a layer of mobile water whose thickness decreases with temperature, leading to drying out of the fibrils.

Hexagonal ice in pure water and biological NMR samples.

Ice, in addition to "liquid" water and protein, is an important component of protein samples for NMR spectroscopy at subfreezing temperatures but it has rarely been observed spectroscopically in this context. We characterize its spectroscopic behavior in the temperature range from 100 to 273 K, and find that it behaves like pure water ice. The interference of magic-angle spinning (MAS) as well as rf multiple-pulse sequences with Bjerrum-defect motion greatly influences the ice spectra.

Solid-state NMR sequential assignment of the ß-endorphin peptide in its amyloid form.

Insights into the three-dimensional structure of hormone fibrils are crucial for a detailed understanding of how an amyloid structure allows the storage of hormones in secretory vesicles prior to hormone secretion into the blood stream. As an example for various hormone amyloids, we have studied the endogenous opioid neuropeptide ß-endorphin in one of its fibril forms. We have achieved the sequential assignment of the chemical shifts of the backbone and side-chain heavy atoms of the fibril. The secondary chemical shift analysis revealed that the ß-endorphin peptide adopts three ß-strands in its fibril state. This finding fosters the amyloid nature of a hormone at the atomic level.

Further exploration of the conformational space of α-synuclein fibrils: solid-state NMR assignment of a high-pH polymorph.

Polymorphism is a common and important phenomenon for protein fibrils which has been linked to the appearance of strains in prion and other neurodegenerative diseases. Parkinson disease is a frequently occurring neurodegenerative pathology, tightly associated with the formation of Lewy bodies. These deposits mainly consist of α-synuclein in fibrillar, ß-sheet-rich form. α-synuclein is known to form numerous different polymorphs, which show distinct structural features. Here, we describe the chemical shift assignments, and derive the secondary structure, of a polymorph that was fibrillized at higher-than-physiological pH conditions. The fibrillar core contains residues 40-95, with both the C- and N-terminus not showing any ordered, rigid parts. The chemical shifts are similar to those recorded previously for an assigned polymorph that was fibrillized at neutral pH.

Dynamic Assembly and Disassembly of Functional ß-Endorphin Amyloid Fibrils.

Neuropeptides and peptide hormones are stored in the amyloid state in dense-core vesicles of secretory cells. Secreted peptides experience dramatic environmental changes in the secretory pathway, from the endoplasmic reticulum via secretory vesicles to release into the interstitial space or blood. The molecular mechanisms of amyloid formation during packing of peptides into secretory vesicles and amyloid dissociation upon release remain unknown. In the present work, we applied thioflavin T binding, tyrosine intrinsic fluorescence, fluorescence anisotropy measurements, and solid-state NMR spectroscopy to study the influence of physiologically relevant environmental factors on the assembly and disassembly of ß-endorphin amyloids in vitro. We found that ß-endorphin aggregation and dissociation occur in vitro on relatively short time scales, comparable to times required for protein synthesis and the rise of peptide concentration in the blood, respectively. Both assembly and disassembly of amyloids strongly depend on the presence of salts of polyprotic acids (such as phosphate and sulfate), while salts of monoprotic acids are not effective in promoting aggregation. A steep increase of the peptide aggregation rate constant upon increase of solution pH from 5.0 to 6.0 toward the isoelectric point as well as more rapid dissociation of ß-endorphin amyloid fibrils at lower pH indicate the contribution of ion-specific effects into dynamics of the amyloid. Several low-molecular-weight carbohydrates exhibit the same effect on ß-endorphin aggregation as phosphate. Moreover, no structural difference was detected between the phosphate- and carbohydrate-induced fibrils by solid-state NMR. In contrast, ß-endorphin amyloid fibrils obtained in the presence of heparin demonstrated distinctly different behavior, which we attributed to a dramatic change of the amyloid structure. Overall, the presented results support the hypothesis that packing of peptide hormones/neuropeptides in dense-core vesicles do not necessarily require a specialized cellular machinery.

Unlike twins: an NMR comparison of two α-synuclein polymorphs featuring different toxicity.

We structurally compare, using solid-state NMR, two different polymorphs of α-synuclein which, as established recently, display contrasting biochemical properties, toxicity, and tropism for cells. We show that both forms, which can each be produced as a pure polymorph, are greatly different in secondary structure. While ß-sheets are the dominating secondary structure elements for both polymorphs, they are markedly divergent in terms of number of elements, as well as their distribution. We demonstrate that all identified ß-sheets feature an in-register parallel stacking for both polymorphs. The two forms show a different molecular arrangement in the unit cell and distinct dynamic features, while sharing a highly flexible C-terminal domain. The use of reproducible, well-identified conditions for sample preparation and the recording of identical NMR experiments allows for a direct comparison of the results.

Yet another polymorph of α-synuclein: solid-state sequential assignments.

Parkinson's disease is a neurological human proteinopathy, which is caused by the accumulation of protein aggregates of high molecular mass. α-Synuclein is a major component of these fibrillar, ß-sheet rich, insoluble assemblies and is deposited in the form of amyloids. Structural characterization of amyloids is possible by solid-state NMR, although no atomic-resolution structure is available as of today. α-Synuclein, as many other pathology-related fibril-forming proteins, can form a number of different polymorphs that are sometimes tricky to obtain in pure form. Here, we describe the chemical shifts and secondary structure analysis of a polymorph that also adopts mainly ß-sheet conformation, with a fibrillar core ranging from residues 38 to 94. In addition, residues 15-20 from the N-terminus found to be part of a rigid ordered ß-sheet. The chemical shifts differ substantially from the polymorph we previously assigned.

Structural and functional characterization of two alpha-synuclein strains.

α-Synuclein aggregation is implicated in a variety of diseases including Parkinson's disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.

Automated solid-state NMR resonance assignment of protein microcrystals and amyloids.

Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218-289) and α-synuclein yielded 88-97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77-90 % correctness if also assignments classified as tentative by the algorithm are included.

Simultaneous acquisition of PAR and PAIN spectra.

We present a scheme that allows the simultaneous detection of PAR and PAIN correlation spectra in a single two-dimensional experiment. For both spectra, we obtain almost the same signal-to-noise ratio as if a PAR or PAIN spectrum is recorded separately, which in turn implies that one of the spectra may be considered additional information for free. The experiment is based on the observation that in a PAIN experiment, the PAR condition is always also fulfilled. The performance is demonstrated experimentally using uniformly (13)C,(15)N-labeled samples of N-f-MLF-OH and ubiquitin.

Solid-state NMR sequential assignments of α-synuclein.

Parkinson's disease is amongst the most frequent and most devastating neurodegenerative diseases. It is tightly associated with the assembly of proteins into high-molecular weight protein species, which propagate between neurons in the central nervous system. The principal protein involved in this process is α-synuclein which is a structural component of the Lewy bodies observed in diseased brain. We here present the solid-state NMR sequential assignments of a new fibrillar form of this protein, the first one with a well-ordered and rigid N-terminal part.

Probing water accessibility in HET-s(218-289) amyloid fibrils by solid-state NMR.

Despite the importance of protein fibrils in the context of conformational diseases, information on their structure is still sparse. Hydrogen/deuterium exchange measurements of backbone amide protons allow the identification hydrogen-bonding patterns and reveal pertinent information on the amyloid ß-sheet architecture. However, they provide only little information on the identity of residues exposed to solvent or buried inside the fibril core. NMR spectroscopy is a potent method for identifying solvent-accessible residues in proteins via observation of polarization transfer between chemically exchanging side-chain protons and water protons. We show here that the combined use of highly deuterated samples and fast magic-angle spinning greatly attenuates unwanted spin diffusion and allows identification of polarization exchange with the solvent in a site-specific manner. We apply this measurement protocol to HET-s(218-289) prion fibrils under different conditions (including physiological pH, where protofibrils assemble together into thicker fibrils) and demonstrate that each protofibril of HET-s(218-289), is surrounded by water, thus excluding the existence of extended dry interfibril contacts. We also show that exchangeable side-chain protons inside the hydrophobic core of HET-s(218-289) do not exchange over time intervals of weeks to months. The experiments proposed in this study can provide insight into the detailed structural features of amyloid fibrils in general.

Motional heterogeneity in single-site silica-supported species revealed by deuteron NMR.

The molecular dynamics of [-SiDMe(2)] grafted on two amorphous silica materials, mesoporous SBA and non-porous Aerosil, was investigated by deuteron ((2)H) solid-state NMR spectroscopy. Quadrupole echo (QE), quadrupole Carr-Purcell-Meiboom-Gill (QCPMG) and magic angle spinning (MAS) spectra were recorded as a function of temperature. These were analyzed to determine the rates and trajectories of molecular motion of the surface species. The dynamics were modelled as a composite two frame motion with independent rotations around the two Si-O bonds. In the first frame there are fast three-site jumps of the -SiDMe(2) group described by a single rate (k(1)) and unequal populations of the tetrahedral sites, such that the ratio D : Me : Me is around 1 : 4 : 4. In the second frame, the Si-O axis makes small step, nearest-neighbour jumps at a rate k(2) along an arc defined by the rim of a cone with a fixed half-angle. Both rates were found to be in the fast motional regime (k(1,2) > 10(10) s(-1)) throughout the experimentally accessible temperature range, 190-350 K. The experimental data are compatible only with models that include a distribution of arc lengths, lambda, in the second frame. The best fit of the simulations to the experimental data yields the distributions of the arc length. The results unequivocally demonstrate that even though the sites all have the same average environment, as reported by the isotropic chemical shifts, the dynamics of the grafted species are microscopically spatially heterogeneous with different molecules on the surface having different ranges of motional trajectories and populations. Furthermore, a clear difference in dynamic behavior is observed between the two silica supports, the motion being more constrained on the mesoporous SBA. This differential mobility is possibly due to differences in surface roughness and to the pore structure of SBA compared with the smoother surface of Aerosil.