Recommendations for addressing the translational gap between experimental and clinical research on amyloid diseases.

This paper is a report of recommendations for addressing translational challenges in amyloid disease research. They were developed during and following an international online workshop organized by the LINXS Institute of Advanced Neutron and X-Ray Science in March 2021. Key suggestions include improving cross-cultural communication between basic science and clinical research, increasing the influence of scientific societies and journals (vis-à-vis funding agencies and pharmaceutical companies), improving the dissemination of negative results, and strengthening the ethos of science.

Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation.

Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII-membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII-lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC-SM-Chol membranes in a manner identical to that of the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein-membrane interactions available from neutron reflection measurements.

The aggregation of "native" human serum albumin.

Recombinant fully deuterated, defatted human serum albumin in heavy water was found to be about 90% aggregated before final fractionation. For comparison and to establish a datum for this isotope effect, the extent of aggregation is reported for "native" defatted and fatted human serum albumin solutions in phosphate buffered 1 mg/ml in heavy and light water at 25 °C and at 4 °C. The extent of aggregation is small over a month at these temperatures, but extensive when the solutions are subjected to repeated freeze-thawing from -18 to 25 °C in both D2O and H2O.

Using neutron protein crystallography to understand enzyme mechanisms.

A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated D-xylose isomerase. These represent four stages in the progression of the biochemical catalytic action of this enzyme. Analyses of the structural changes observed between the various three-dimensional structures lead to some insight into the mechanism of action of this enzyme.

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 ß-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids.

Water-DNA interactions as studied by X-ray and neutron fibre diffraction.

X-ray fibre-diffraction studies indicate a high degree of stereochemical specificity in interactions between water and the DNA double helix. Evidence for this comes from data that show that the molecular conformations assumed by DNA in fibres are highly reproducible and that the hydration-driven transitions between these conformations are fully reversible. These conformational transitions are induced by varying the relative humidity of the fibre environment and hence its water content. Further evidence for stereochemical specificity comes from the observed dependence of the conformation assumed on the ionic content of the fibre and the nucleotide sequence of the DNA. For some transitions, information on stereochemical pathways has come from real-time X-ray fibre diffraction using synchrotron radiation; information on the location of water with respect to the double helix for a number of DNA conformations has come from neutron fibre diffraction. This structural information from fibre-diffraction studies of DNA is complemented by information from X-ray single-crystal studies of oligonucleotides. If the biochemical processes involving DNA have evolved to exploit the structural features observed in DNA fibres and oligonucleotide single crystals, the challenges in developing alternatives to a water environment can be expected to be very severe.