Trevor Forsyth

A high-angle neutron fibre diffraction study of the hydration of deuterated A-DNA

A high-angle neutron fibre diffraction study of the hydration of A-DNA has been performed using the single-crystal diffractometer D19 at the Institut Laue-Langevin (Grenoble, France). The sample was prepared using deuterated DNA extracted from E. Coli cells cultured on deuterated nutrients. In common with our previous neutron fibre diffraction studies of DNA, this work exploits the ability to isotopically replace H2O around the DNA by D2O. However this study benefitted additionally from the fact that the hydrogen atoms which are covalently bonded to carbon atoms in the DNA sugars and bases were replaced by deuterium so that incoherent scattering and absorption effects were minimised. Successive cycles of Fourier synthesis and Fourier difference synthesis allowed water peaks to be identified and their positional and occupancy parameters to be refined against the observed diffraction data. The results confirm the main hydration features noted in our earlier studies with a clear network of water running along the inside edge of the major groove linking successive OI phosphate oxygen atoms. The central core of water running along the axis of the double helix is very much clearer in this work. Additionally this study shows chains of ordered water lying in the centre of the major groove.

THE STRUCTURE OF CELLULOSES

X-ray and neutron fiber diffraction has been used to study cellulose as it is converted from its naturally occurring crystal phase, cellulose I, to an activated crystal phase, cellulose IIII, by ammonia treatment. The detailed crystal structures determined for cellulose Iβ, an intermediate ammonia-cellulose I complex, and cellulose IIII, reveal a structural transition pathway: hydrogen bonded sheets of chains in cellulose Iβ slip with respect to each other to accommodate the penetrating ammonia guest molecules in the intermediate complex. On evaporation of ammonia from the intermediate complex, there is a relative small change in chain packing as an inter-sheet ammonia bridge is replaced by an inter-sheet hydrogen bond in cellulose IIII. When cellulose IIII is heated it converts back to cellulose Iβ. Both ammonia-cellulose I and cellulose IIII have extended chains of cooperative hydrogen bonds in relative open crystal structures that may add to their susceptibility to rapid change.

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.

Structural polymorphism in a tubercidin analogue of the DNA double helix

A high-angle X-ray fibre diffraction study of a tubercidin analogue of the poly[d(A-T)].poly[d(A-T)] DNA double helix has been carried out using station 7.2 at the Daresbury Laboratory synchrotron radiation source. The polymer has been studied for a wide range of salt strengths and hydration conditions and exhibits conformational polymorphism that is quite distinct from that observed for the unmodified polymer. The replacement of deoxyadenosine by deoxytubercidin in the polynucleotide causes only slight alterations to the structure of A-DNA, but significantly alters the structure of the B conformation. Additionally, the modified polymer does not, in any conditions yet identified, adopt the D conformation. In conditions which would normally favour the D conformation of poly[d(A-T)].poly[d(A-T)], the modified polymer adopts an unusual conformation which is designated here as the K conformation. These observations are important for an understanding of major groove interactions involved in the stabilisation of particular DNA conformations and also more generally for an insight into the pharmacological activity of tubercidin which following its incorporation into nucleic acids may cause stereochemical distortions of the DNA double helix.

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 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.

Characterising PC/cholesterol supported lipid bilayers and interactions with human HDL

Sarah Hannah Anne Waldie1, Kathryn Browning2, Martine Moulin3, Michael Haertlein3, Trevor Forsyth3, Selma Maric4, Marité Cárdenas4 1Department Of Biomedical Science, Malmo University/Life Sciences Group, ILL, Grenoble, France, 2Department of Pharmacy, Uppsala University, Uppsala, Sweden, 3Life Sciences Group, ILL, Grenoble, France, 4Department of Biomedical Science, Malmo University, Malmo, Sweden E-mail: waldies@ill.fr

Structural studies of dynamic CD4 changes relevant to HIV infection

Cluster of differentiation 4 (CD4) plays an important role in the adaptive immune response. It acts co-operatively with the T-cell receptor (TCR) to bind the Major Histocompatibility Complex Class 2 (MHC2) on antigen-presenting cells (APCs) to effect down-stream immune responses. However, it is also the primary receptor for the Human Immunodeficiency Virus-1 (HIV-1) which binds to human CD4+ cells via the surface glycoprotein gp120. CD4 has 4 ectodomains (D1-4) of which domains D1, D2 and D4 contain disulphide bonds. The second domain disulphide bond is classed as an allosteric disulphide, of the configuration “–RHStaple”, which has unusually high dihedral energy, resulting in its relatively facile reduction and potential structural realignment.

Unravelling transthyretin amyloidosis by neutron crystallography

Ai Woon Yee1, Martine Moulin1, Matthew Blakeley2, Jonathan Cooper3, Michael Haertlein1, Edward Mitchell4, Trevor Forsyth1 1Life Sciences Group, Institut Laue-Langevin, Grenoble, France, 2Large-Scale Structures Group, Institut Laue-Langevin, Grenoble, France, 3Laboratory of Protein Crystallography, Drug Discovery Group, Wolfson Institute for Biomedical Research, UCL, London, United Kingdom, 4Business Development Unit, European Synchrotron Radiation Facility (ESRF), Grenoble, France E-mail: yee@ill.fr

Targeting transthyretin amyloidosis: Joint neutron and X-ray diffraction analysis of a pathogenic protein

Transthyretin (TTR) amyloidosis is the most common hereditary form of amyloidosis that is characterised by extracellular deposition of insoluble amyloid fibrils derived from misfolded protein in one or more organ systems in the body.1 It is an irreversible and progressive disease and is fatal within 10 years of onset. Autosomal dominant mutations2 in the TTR gene alter the protein stability leading to tetramer dissociation and favouring an abnormal monomeric structure, which in turn polymerises into unknown intermediates and finally into amyloid fibrils.3 Up to date, more than 200 X-ray crystal structures are available for TTR, but there is no consistent model to conclude the molecular assembly of the fibril building blocks and the triggering factors for this process. Neutron protein crystallography is a powerful tool that strongly complements X-ray structural studies by revealing key details of hydrogen atom interactions within the protein. Looking at the differences in hydrogen bonding, protonation states and hydration of two TTR mutants – S52P and T119M, which play opposite roles in the protein stability,4,5 will provide key insights into how they destabilise the tetramer and promote amyloidotic aggregation. References: 1. Planté-Bordeneuve V, Said G. Lancet Neurol. 2011;10(12):1086-1097. 2. Saraiva MJM. Database on Transthyretin Mutations. 2013:1-24. 3. Damas AM, Saraiva MJ. J Struct Biol. 2000;130(2-3):290-9. 4. Mangione PP, Porcari R, Gillmore JD, et al. Proc Natl Acad Sci U S A. 2014;111(4):1539-44. 5. Sebastião MP, Lamzin V, Saraiva MJ, Damas a M. J Mol Biol. 2001;306:733-44.