Carolin Seuring

Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein

Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal. Structural changes during a macromolecular reaction are captured at near-atomic resolution by an x-ray free electron laser. Watching a protein molecule in motion X-ray crystallography has yielded beautiful high-resolution images that give insight into how proteins function. However, these represent static snapshots of what are often dynamic processes. For photosensitive molecules, time-resolved crystallography at a traditional synchrotron source provides a method to follow structural changes with a time resolution of about 100 ps. X-ray free electron lasers (XFELs) open the possibility of performing time-resolved experiments on time scales as short as femtoseconds. Tenboer et al. used XFELs to study the light-triggered dynamics of photoactive yellow protein. Electron density maps of high quality were obtained 10 ns and 1 µs after initiating the reaction. At 1 µs, two intermediates revealed previously unidentified structural changes. Science, this issue p. 1242

The Mechanism of Toxicity in HET-S/HET-s Prion Incompatibility

A nontoxic functional prion activates toxicity in the HET-S/HET-s fungal heterokaryon incompatibility system by converting HET-S into a cytotoxic membrane protein.

Dynamic Assembly and Disassembly of Functional β-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 β-endorphin amyloids in vitro. We found that β-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 β-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 β-endorphin aggregation as phosphate. Moreover, no structural difference was detected between the phosphate- and carbohydrate-induced fibrils by solid-state NMR. In contrast, β-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.

Mix-and-diffuse serial synchrotron crystallography

The structure of chitotriose bound to lysozyme after mixing times of 2 and 50 s was determined using a polyimide tape-drive device for mix-and-diffuse serial crystallography at a synchrotron light source.

Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different

Amyloid polymorphism of twisted and straight β-endorphin fibrils was studied by negative-stain transmission electron microscopy, scanning transmission electron microscopy, and solid-state nuclear magnetic resonance spectroscopy. Whereas fibrils assembled in the presence of salt formed flat, striated ribbons, in the absence of salt they formed mainly twisted filaments. To get insights into their structural differences at the atomic level, 3D solid-state NMR spectra of both fibril types were acquired, allowing the detection of the differences in chemical shifts of 13C and 15N atoms in both preparations. The spectral fingerprints and therefore the chemical shifts are very similar for both fibril types. This indicates that the monomer structure and the molecular interfaces are almost the same but that these small differences do propagate to produce flat and twisted morphologies at the mesoscopic scale. This finding is in agreement with both experimental and theoretical considerations on the assembly of polymers (including amyloids) under different salt conditions, which attribute the mesoscopic difference of flat versus twisted fibrils to electrostatic intermolecular repulsions.

Analysis of XFEL serial diffraction data from individual crystalline fibrils

Methods are described for processing XFEL data from individual crystalline fibrils. The methods are applied to data collected at the Linac Coherent Light Source from an amyloid-forming oligopeptide from the adenovirus shaft.

Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser.

A major goal for X‐ray free‐electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one‐dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F‐actin, and amyloid fibrils), which when intersected by femtosecond X‐ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F‐actin can be flow‐aligned to a disorientation of approximately 5 degrees. Using this XFEL‐based technique, we determine that gelsolin amyloids are comprised of stacked β‐strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α‐synuclein amyloids.

Solid-state NMR sequential assignment of the β-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 β-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 β-endorphin peptide adopts three β-strands in its fibril state. This finding fosters the amyloid nature of a hormone at the atomic level.

X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography

Multi-crystal serial crystallography data can be used for UV and X-ray radiation-damage-induced phasing.

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.The structures of amyloid fibres are currently primarily studied through solid state NMR and cryo-EM. Here the authors present a free-standing graphene support device that allows diffraction imaging of non-crystalline amyloid fibrils with single X-ray pulses from an X-ray free-electron laser.