X-ray Fiber Diffraction Studies Research Articles

X-Ray Fiber Diffraction and Numerical Simulation Studies on the Change in the Helical Symmetry of Chlamydomonas Flagellar Axonemes Coupled with the Change in Ca2+ Concentrations

Cilia and flagella are motile organelles found in various types of eukaryotes. In their axonemes, dynein arms generate shear between adjacent doublets, and this shear is converted into complex waveforms. The conversion mechanism has remained a long-standing unresolved issue. Since Chlamydomonas flagella change their waveforms from a flagellar type to a ciliary type, responding to the increase in intracellular Ca2+ concentrations, this response may provide a hint for the mechanism of the flagellar waveform generation.

Hydration forces between aligned DNA helices undergoing B to A conformational change: In-situ X-ray fiber diffraction studies in a humidity and temperature controlled environment.

Hydration forces between DNA molecules in the A- and B-Form were studied using a newly developed technique enabling simultaneous in situ control of temperature and relative humidity. X-ray diffraction data were collected from oriented calf-thymus DNA fibers in the relative humidity range of 98%-70%, during which DNA undergoes the B- to A-form transition. Coexistence of both forms was observed over a finite humidity range at the transition. The change in DNA separation in response to variation in humidity, i.e. change of chemical potential, led to the derivation of a force-distance curve with a characteristic exponential decay constant of∼2Å for both A- and B-DNA. While previous osmotic stress measurements had yielded similar force-decay constants, they were limited to B-DNA with a surface separation (wall-to-wall distance) typically>5Å. The current investigation confirms that the hydration force remains dominant even in the dry A-DNA state and at surface separation down to∼1.5Å, within the first hydration shell. It is shown that the observed chemical potential difference between the A and B states could be attributed to the water layer inside the major and minor grooves of the A-DNA double helices, which can partially interpenetrate each other in the tightly packed A phase. The humidity-controlled X-ray diffraction method described here can be employed to perform direct force measurements on a broad range of biological structures such as membranes and filamentous protein networks.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells.

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

On the Helical Structure of Guanosine 5'-Monophosphate Formed at pH 5: Is It Left- or Right-Handed?

Early X-ray fiber diffraction studies have established that the spontaneous gel formation of guanosine 5′-monophosphate (5′-GMP) under slightly acidic conditions (e.g., pH 5) results from self-assembly of 5′-GMP into a helical structure in which hydrogen-bonded guanine bases form a continuous helix with 15 nucleotides per 4 turns. For more than five decades, the sense of this helix is believed to be left-handed. Using multinuclear solid-state NMR and IR spectroscopic methods, we have finally determined the long-missing structural details of this helix. First, we found that this 5′-GMP helix is right-handed containing exclusive C3′-endo sugar puckers. Second, we showed that the central channel of this helix is free of Na+ ions, which is in sharp contrast to the helix formed by 5′-GMP at pH 8 where the central channel is filled with Na+ ions.

Cocrystalline Copolyimides of Poly(ethylene 2,6-naphthalate).

Copolycondensation of N,N'-bis(2-hydroxyethyl)-biphenyl-3,4,3',4'-tetracarboxylic diimide (5-25 mol %) with bis(2-hydroxyethyl)-2,6-naphthalate affords a series of cocrystalline, poly(ethylene 2,6-naphthalate) (PEN)-based poly(ester imide)s. The glass transition temperature rises with the level of comonomer, from 118 °C for PEN itself to 148 °C for the 25% diimide copolymer. X-ray powder and fiber diffraction studies show that, when 5 mol % or more of diimide is present, the α-PEN crystal structure is replaced by a new crystalline phase arising from isomorphic substitution of biphenyldiimide for PEN residues in the polymer crystal lattice. This new phase is provisionally identified as monoclinic, C2/m, with two chains per unit cell, a = 10.56, b = 6.74, c = 13.25 Å, and β = 143.0°.

Fifty years of fibrous protein research: A personal retrospective

As a result of X-ray fiber diffraction studies on fibrous proteins and crystallographic data on fragments derived from them, new experimental techniques across the biophysical and biochemical spectra, sophisticated computer modeling and refinement procedures, widespread use of bioinformatics and improved specimen preparative procedures the structures of many fibrous proteins have now been determined to at least low resolution. In so doing these structures have yielded insight into the relationship that exists between sequence and conformation and this, in turn, has led to improved methodologies for predicting structure from sequence data alone. In this personal retrospective a selection of progress made during the past 50years is discussed in terms of events to which the author has made some contribution.

Spider Glue Proteins Have Distinct Architectures Compared with Traditional Spidroin Family Members

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.

Inhibition of interdomain motion in g‐actin by the natural product latrunculin: A molecular dynamics study

As part of the cytoskeleton, actin is essential for the morphology, motility, and division of eukaryotic cells. Recent X-ray fiber diffraction studies have shown that the conformation of monomeric actin is flattened upon incorporation into the filament by a relative rotation of its two major domains. The antiproliferative activity of latrunculin, a macrolide toxin produced by sponges, seems to be related to its binding to monomeric actin and inhibition of polymerization. Yet, the mechanism of inhibition is not known in detail. Here, multiple explicit water molecular dynamics simulations show that latrunculin binding hinders the conformational transition related to actin polymerization. In particular, the presence of latrunculin at the interface of the two major domains of monomeric actin reduces the correlated displacement of Domain 2 with respect to Domain 1. Moreover, higher rotational flexibility between the two major domains is observed in the absence of ATP as compared to ATP-bound actin, offering a possible explanation as to why actin polymerizes more favorably in the absence of nucleotides.

Neutron fibre diffraction studies of amyloid using H2O/D2O isotopic replacement.

The first neutron fibre diffraction studies of an amyloid system are presented. The techniques used to prepare the large samples needed are described, as well as the procedures used to isotopically replace H2O in the sample by D2O. The results demonstrate the feasibility of this type of approach for the pursuit of novel structural analyses that will strongly complement X-ray fibre diffraction studies and probe aspects of amyloid structure that to date have remained obscure. The approach is demonstrated using an amyloid form of the peptide NSGAITIG, but is equally applicable for the study of other systems such as Alzheimer's Aβ peptide.

Methionine oxidation induces amyloid fibril formation by full-length apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (T(m)) from 58.7 degrees C for native apoA-I to 48.2 degrees C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 A and 9.92 A, indicating a cross-beta amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation.

Synchrotron X-ray fiber diffraction study on the thermal expansion behavior of cellulose crystals in tension wood of Japanese poplar in the low-temperature region

Abstract The thermal expansion behavior of tension wood cellulose obtained from Populus maximowiczii A. Henry was investigated by synchrotron X-ray fiber diffraction at temperatures ranging from 100 to 300 K. Three equatorial and one meridional d-spacings indexed as , 110, 200, and 004 showed a gradual linear increase with increasing temperature. Unit-cell parameters calculated from the changes in these d-spacings showed anisotropic thermal expansion behavior in the three axial directions related to the crystal structure and hydrogen-bonding system of cellulose Iβ. The linear thermal expansion coefficients (TECs) of the a-, b-, and c-axes were: α a=5.2×10-5 K-1, α b=2.1×10-5 K-1, and α c=0.4×10-5 K-1, respectively. The volume TEC was β=7.8×10-5 K-1, which was approximately 70% of previously reported values in the high-temperature region from room temperature to 250°C.

The Structure of a Filamentous Bacteriophage

Many thin helical polymers, including bacterial pili and filamentous bacteriophage, have been seen as refractory to high-resolution studies by electron microscopy. Studies of the quaternary structure of such filaments have depended upon techniques such as modeling or X-ray fiber diffraction, given that direct visualization of the subunit organization has not been possible. We report the first image reconstruction of a filamentous virus, bacteriophage fd, by cryoelectron microscopy. Although these thin (∼ 70 Å in diameter) rather featureless filaments scatter weakly, we have been able to achieve a nominal resolution of ∼8 Å using an iterative helical reconstruction procedure. We show that two different conformations of the virus exist, and that in both states the subunits are packed differently than in conflicting models previously proposed on the basis of X-ray fiber diffraction or solid-state NMR studies. A significant fraction of the population of wild-type fd is either disordered or in multiple conformational states, while in the presence of the Y21M mutation, this heterogeneity is greatly reduced, consistent with previous observations. These results show that new computational approaches to helical reconstruction can greatly extend the ability to visualize heterogeneous protein polymers at a reasonably high resolution.

Molecular Structure of fd (f1, M13) Filamentous Bacteriophage Refined with Respect to X-ray Fibre Diffraction and Solid-state NMR Data Supports Specific Models of Phage Assembly at the Bacterial Membrane

Filamentous bacteriophage (Inovirus) is a simple and well-characterized model system. The phage particle, or virion, is about 60 angstroms in diameter and several thousand angstrom units long. The virions are assembled at the bacterial membrane as they extrude out of the host without killing it, an example of specific transport of nucleoprotein assemblages across membranes. The Ff group (fd, f1 and M13) has been especially widely studied. Models of virion assembly have been proposed based on a molecular model of the fd virion derived by X-ray fibre diffraction. A somewhat different model of the fd virion using solid-state NMR data has been proposed, not consistent with these models of assembly nor with the X-ray diffraction data. Here we show that reinterpreted NMR data are also consistent with the model derived from X-ray fibre diffraction studies, and discuss models of virion assembly.

Polyglutamine homopolymers having 8–45 residues form slablike β‐crystallite assemblies

At least nine inherited neurodegenerative diseases, including Huntington's, are caused by poly(L-glutamine) (polyGln, polyQ) expansions > 35-40 repeats in widely or ubiquitously expressed proteins. Except for their expansions, these proteins have no sequence homologies, and their functions mostly remain unknown. Although each disease is characterized by a distinct pathology specific to a subset of neuronal cells, the formation of neuronal intranuclear aggregates containing protein with an expanded polyQ is the hallmark and common feature to most polyQ disorders. The neurodegeneration is thought to be caused by a toxic gain of function that occurs at the protein level and depends on the length of the expansion: Longer repeats cause earlier age of onset and more severe symptoms. To address whether there is a structural difference between polyQ having < 40 versus > 40 residues, we undertook an X-ray fiber diffraction study of synthetic polyQ peptides having varying numbers of residues: Ac-Q8-NH2, D2Q15K2, K2Q28K2, and K2Q45K2. These particular lengths bracket both the range of normalcy (9-36 repeats) and the pathological (45 repeats), and therefore could be indicative of the structural changes expected in expanded polyQ domains. Contrary to expectations of different length-dependent morphologies, we accounted for all the X-ray patterns by slablike, beta-sheet structures, approximately 20 A thick in the beta-chain direction, all having similar monoclinic lattices. Moreover, the slab thickness indicates that K2Q45K2, rather than forming a water-filled nanotube, must form multiple reverse turns.

Combined X-ray and neutron fibre diffraction studies of biological and synthetic polymers

The fibrous state is a natural one for polymer molecules which tend to assume regular helical conformations rather than the globular structures characteristic of many proteins. Fibre diffraction therefore has broad application to the study of a wide range of biological and synthetic polymers. The purpose of this paper is to illustrate the general scope of the method and in particular to demonstrate the impact of a combined approach involving both X-ray and neutron diffraction methods.While the flux of modern X-ray synchrotron radiation sources allows high quality datasets to be recorded with good resolution within a very short space of time, neutron studies can provide unique information through the ability to locate hydrogen or deuterium atoms that are often difficult or impossible to locate using X-ray methods. Furthermore, neutron fibre diffraction methods can, through the ability to selectively label specific parts of a structure, be used to highlight novel aspects of polymer structure that can not be studied using X-rays.Two examples are given. The first describes X-ray and neutron diffraction studies of conformational transitions in DNA. The second describes structural studies of the synthetic high-performance polymer poly(p-phenylene terephthalamide) (PPTA), known commercially as Kevlar® or Twaron®.

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.

Fibrillin-rich microfibrils: elastic biopolymers of the extracellular matrix.

Fibrillin-rich microfibrils are evolutionarily ancient macromolecular assemblies of the extracellular matrix. They have unique extensible properties that endow vascular and other tissues with long-range elasticity. Microfibril extensibility supports the low pressure closed circulations of lower organisms such as crustaceans. In higher vertebrates, microfibrils act as a template for elastin deposition and are components of mature elastic fibres. In man, the importance of microfibrils is highlighted by the linkage of mutations in their principal structural component, fibrillin-1, to the heritable disease Marfan syndrome which is characterised by severe cardiovascular, skeletal and ocular defects. When isolated from tissues, fibrillin-rich microfibrils have a complex ultrastructural organisation with a characteristic 'beads-on-a-strong' appearance. X-ray fibre diffraction studies and biomechanical testing have shown that microfibrils are reversibly extensible at tissue extensions of 100%. Ultrastructural analysis and 3D reconstructions of isolated microfibrils using automated electron tomography have revealed new details of how fibrillin molecules are aligned within microfibrils in untensioned and extended states, and delineated the role of calcium in regulating microfibril beaded periodicity, rest length and molecular organisation. The molecular basis of how fibrillin molecules assemble into microfibrils, the central role of cells in regulating this process, and the identity of other molecules that may coassemble into microfibrils are now being elucidated. This information will enhance our understanding of the elastic mechanism of these unique extracellular matrix polymers, and may lead to new microfibril-based strategies for repairing elastic tissues in ageing and disease.

Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils reveals interstrand contact regions between assembled beta-amyloid peptide subunits.

The assembly of the beta-amyloid peptide (Abeta) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural information about fibrillogenesis has remained elusive due to the highly insoluble, noncrystalline nature of the assembled peptide. X-ray fiber diffraction, infrared spectroscopy, and solid-state NMR studies performed on fibrils composed of Abeta peptides have led to conflicting models of the intermolecular alignment of beta-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Abeta monomers within amyloid fibrils. A photoreactive Abeta(1-40) ligand was synthesized by substituting L-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Abeta(1-40) F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS-PAGE of dissolved fibrils revealed the light-dependent formation of a covalent Abeta dimer. Enzymatic cleavage followed by mass spectrometric analysis demonstrated the presence of a dimer-specific ion at MH(+) = 1825.9, the predicted mass of a fragment composed of the N-terminal Abeta(1-5) F4Bpa tryptic peptide covalently attached to the C-terminal Abeta(29-40) tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked dimer led to the localization of the photo-cross-link between the ketone of the Bpa4 side chain and the delta-methyl group of the Met35 side chain. The Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel alignment of Abeta peptides within amyloid fibrils.

A Revised Structure and Hydrogen-Bonding System in Cellulose II from a Neutron Fiber Diffraction Analysis

The crystal and molecular structure and hydrogen bonding system in cellulose II have been revised using new neutron diffraction data extending to 1.2 Å resolution collected from two highly crystalline fiber samples of mercerized flax. Mercerization was achieved in NaOH/H2O for one sample and in NaOD/D2O for the other, corresponding to the labile hydroxymethyl moieties being hydrogenated and deuterated, respectively. Fourier difference maps were calculated in which neutron difference amplitudes were combined with phases calculated from two revised X-ray models of cellulose II, A and B‘. The revised phasing models were determined by refinement against the X-ray data set of Kolpak and Blackwell,8 using the LALS methodology.37 Both models A and B‘ have two antiparallel chains organized in a P21 space group and unit cell parameters: a = 8.01 Å, b = 9.04 Å, c = 10.36 Å, and γ = 117.1°.15 Model A has equivalent backbone conformations for both chains but different conformations for the hydroxymethyl moieties: gt for the origin chain and tg for the center chain. Model B‘, based on the recent crystal structures of cellotetraose,21-23 has different conformations for the two chains but nearly equivalent conformations for the hydroxymethyl moieties. On the basis of the X-ray data alone, model A and model B‘ could not be differentiated. From the neutron Fourier difference maps, possible labile hydrogen atom positions were identified for each model and refined using LALS. We were able to eliminate model A in favor of model B‘. The hydrogen-bonding scheme identified for model B‘ is significantly different from previous proposals based on the crystal structures of cellotetraose,21-23 MD simulations of cellulose II,25 and any potential hydrogen-bonding network in the structure of cellulose II determined in earlier X-ray fiber diffraction studies.7,8 The exact localization of the labile hydrogen atoms involved in this bonding, together with their donor and acceptor characteristics, is presented and discussed. This study provides, for the first time, the coordinates of all of the atoms in cellulose II.

Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing.

Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer's disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self-assemble to form insoluble fibrils. X-ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross-beta-scaffold, with beta-strands perpendicular and beta-sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol-3'-kinase, using cryo-electron microscopy and image processing at 25 A resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of approximately 600 A and an axial subunit repeat of approximately 27 A. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20x40-A protofilaments can only accommodate one pair of flat beta-sheets stacked against each other, with very little inter-strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.