X-ray Fiber Diffraction Analysis Research Articles

Identifying the Coiled-Coil Triple Helix Structure of β-Peptide Nanofibers at Atomic Resolution

Peptide self-assembly represents a powerful bottom-up approach to the fabrication of nanomaterials. β3-Peptides are non-natural peptides composed entirely of β-amino acids, which have an extra methylene in the backbone, and we reported fibers derived from the self-assembly of β3-peptides that adopt 14-helical structures. β3-Peptide assemblies represent a class of stable nanomaterials that can be used to generate bio- and magneto-responsive materials with proteolytic stability. However, the three-dimensional structure of many of these materials remains unknown. To develop structure-based criteria for the design of β3-peptide-based biomaterials with tailored function, we investigated the structure of a tri-β3-peptide nanoassembly by molecular dynamics simulations and X-ray fiber diffraction analysis. Diffraction data was collected from aligned fibrils formed by Ac-β3[LIA] in water and used to inform and validate the model structure. Models with 3-fold radial symmetry resulted in stable fibers with a triple-helical coiled-coil motif and measurable helical pitch and periodicity. The fiber models revealed a hydrophobic core and twist along the fiber axis arising from a maximization of contacts between hydrophobic groups of adjacent tripeptides on the solvent-exposed fiber surface. These atomic structures of macroscale fibers derived from β3-peptide-based materials provide valuable insight into the effects of the geometric placement of the side chains and the influence of solvent on the core fiber structure which is perpetuated in the superstructure morphology.

Bacillus subtilis HelD, an RNA Polymerase Interacting Helicase, Forms Amyloid-Like Fibrils.

HelD, an RNA polymerase binding protein from Bacillus subtilis, stimulates transcription and helps in timely adaptation of cells under diverse environmental conditions. At present, no structural information is available for HelD. In the current study, we performed size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS) which suggests that HelD is predominantly monomeric and globular in solution. Using combination of size exclusion chromatography and analytical ultracentrifugation, we also show that HelD has a tendency to form higher order oligomers in solution. CD experiments suggest that HelD has both α-helical (∼35%) and β sheet (∼26%) secondary structural elements. Thermal melting experiments suggest that even at 90°C, there is only about 30% loss in secondary structural contents with Tm of 44°C. However, with the increase in temperature, there was a gain in the β-sheet content and significant irreversible loss of α-helical content. Using a combination of X-ray fiber diffraction analysis, and dye based assays including Thioflavin-T based fluorescence and Congo red binding assays, we discovered that HelD forms amyloid-like fibrils at physiologically relevant conditions in vitro. Using confocal imaging, we further show that HelD forms amyloid inclusions in Escherichia coli. Bioinformatics-based sequence analysis performed using three independent web-based servers suggests that HelD has more than 20 hot-spots spread across the sequence that may aid the formation of amyloid-like fibrils. This discovery adds one more member to the growing list of amyloid or amyloid-like fibril forming cytosolic proteins in bacteria. Future studies aimed at resolving the function of amyloid-like fibrils or amyloid inclusions may help better understand their role, if any, in the bacterial physiology.

Peptides for targeting βB2-crystallin fibrils

Crystallins are a major family of proteins located within the lens of the eye. Cataracts are thought to be due to the formation of insoluble fibrillar aggregates, which are largely composed of proteins from the crystallin family. Today the only cataract treatment that exists is surgery and this can be difficult to access for individuals in the developing world. Development of novel pharmacotherapeutic approaches for the treatment of cataract rests on the specific targeting of these structures. βB2-crystallin, a member of β-crystallin family, is a large component of the crystallin proteins within the lens, and as such was used to form model fibrils in vitro. Peptides were identified, using phage display techniques, that bound to these fibrils with high affinity. Fibrillation of recombinantly expressed human βB2-crystallin was performed in 10% (v/v) trifluoroethanol (TFE) solution (pH 2.0) at various temperatures, and its amyloid-like structure was confirmed using Thioflavin-T (ThT) assay, transmission electron microscopy (TEM), and X-ray fiber diffraction (XRFD) analysis. Affinity of identified phage-displayed peptides were analyzed using enzyme-linked immunosorbent assay (ELISA). Specific binding of a cyclic peptide (CKQFKDTTC) showed the highest affinity, which was confirmed using a competitive inhibition assay.

Might tumor secreted cathepsin proteases leave specific molecular signals in skin, hair and nails years before a cancer becomes clinically apparent?

X-ray fiber diffraction analysis (FDA) of the fibrous macromolecules in hair, nails and skin has been shown to non-invasively diagnose various cancers, at sites remote from the cancer, years before the cancer becomes clinically apparent. The technology is not widely accepted because of reproducibility issues (that can be easily resolved) and lack of an explanation as to how a clinically unapparent tumor can leave molecular “signatures” at remote sites. However, there is evidence that tumor-specific cathepsins (lysosomal proteases) circulate systemically long before a cancer is clinically apparent. As such, we hypothesize that cathepsins, by virtue of their proteolytic activity, impart molecular changes in tissues remote from the primary tumor. These subtle molecular changes, which are specific for various tumors, can be readily detected by FDA of hair, nails and skin. We call for more research in the utility of FDA and tumor specific cathepsins for the early and non-invasive diagnosis of various malignancies.

The phase problem for one-dimensional crystals.

The phase problem for diffraction amplitudes measured from a one-dimensional crystal is examined. In the absence of any a priori information, the solution to this problem is shown to be unique up to a parameterized, low-dimensional set of solutions. Minimal additional a priori information is expected to render the solution unique. The effects of additional information such as positivity, molecular envelope and helical symmetry on uniqueness are characterized. The results are pertinent to structural studies of polymeric and rod-like biomolecular assemblies that form one-dimensional, rather than three-dimensional, crystals. This shows the potential for ab initio phasing of diffraction data from single such assemblies measured using new X-ray free-electron laser sources. Such an approach would circumvent the complicated inversion of cylindrically averaged diffraction that is necessary in traditional X-ray fibre diffraction analysis.

Structure of viruses: a short history

This review is a partially personal account of the discovery of virus structure and its implication for virus function. Although I have endeavored to cover all aspects of structural virology and to acknowledge relevant individuals, I know that I have favored taking examples from my own experience in telling this story. I am anxious to apologize to all those who I might have unintentionally offended by omitting their work. The first knowledge of virus structure was a result of Stanley's studies of tobacco mosaic virus (TMV) and the subsequent X-ray fiber diffraction analysis by Bernal and Fankuchen in the 1930s. At about the same time it became apparent that crystals of small RNA plant and animal viruses could diffract X-rays, demonstrating that viruses must have distinct and unique structures. More advances were made in the 1950s with the realization by Watson and Crick that viruses might have icosahedral symmetry. With the improvement of experimental and computational techniques in the 1970s, it became possible to determine the three-dimensional, near-atomic resolution structures of some small icosahedral plant and animal RNA viruses. It was a great surprise that the protecting capsids of the first virus structures to be determined had the same architecture. The capsid proteins of these viruses all had a 'jelly-roll' fold and, furthermore, the organization of the capsid protein in the virus were similar, suggesting a common ancestral virus from which many of today's viruses have evolved. By this time a more detailed structure of TMV had also been established, but both the architecture and capsid protein fold were quite different to that of the icosahedral viruses. The small icosahedral RNA virus structures were also informative of how and where cellular receptors, anti-viral compounds, and neutralizing antibodies bound to these viruses. However, larger lipid membrane enveloped viruses did not form sufficiently ordered crystals to obtain good X-ray diffraction. Starting in the 1990s, these enveloped viruses were studied by combining cryo-electron microscopy of the whole virus with X-ray crystallography of their protein components. These structures gave information on virus assembly, virus neutralization by antibodies, and virus fusion with and entry into the host cell. The same techniques were also employed in the study of complex bacteriophages that were too large to crystallize. Nevertheless, there still remained many pleomorphic, highly pathogenic viruses that lacked the icosahedral symmetry and homogeneity that had made the earlier structural investigations possible. Currently some of these viruses are starting to be studied by combining X-ray crystallography with cryo-electron tomography.

Hydrophobic, Aromatic, and Electrostatic Interactions Play a Central Role in Amyloid Fibril Formation and Stability

Amyloid-like fibrous crystals formed by the peptide KFFEAAAKKFFE have been previously characterized and provide an ideal model system to examine the importance of specific interactions by introducing specific substitutions. We find that the removal of any phenylalanine residue completely abrogates assembly ability, while charged residues modulate interactions within the structure resulting in alternative fibrillar morphologies. X-ray fiber diffraction analysis reveals that the essential backbone packing of the peptide molecules is maintained, while small changes accommodate differences in side chain size in the variants. We conclude that even very short peptides are adaptable and add to the growing knowledge regarding amyloid polymorphisms. Additionally, this work impacts on our understanding of the importance of residue composition for amyloidogenic peptides, in particular the roles of electrostatic, aromatic, and hydrophobic interactions in amyloid assembly.

Structural Changes of Microtubules During GTP Hydrolysis Revealed by X-Ray Fiber-Diffraction

Microtubule (MT) is one of the most essential cytoskeletal elements in the eukaryote cells, which supports the mitosis, cell architecture and motility as well as the intracellular transportation. Tubulin dimers in a GTP-binding state (GTP-tubulin) are assembled onto MT-ends and make them stable. However, the following slow hydrolysis of GTP to GDP occurring in β-tubulin is assumed to weaken the lateral interactions among protofilaments and MTs in an uncapped state (MT-ends without GTP-tubulin) disassemble and shrink quickly. Such features of dynamic instability would be closely related to various MT functions. In the present study, we tested how the GTP-hydrolysis was related to the structural changes of MTs by X-ray fiber diffraction analysis (BL45XU, SPring-8, λ=0.09 nm). By comparing diffraction peaks between GMPCPP- and GTP-taxol-MT, we found that 8 nm meridional reflection intensity (arrow in the figure) appeared only in GTP-taxol-MTs, which are assumed to consist mostly of GDP-tubulin. It would reflect some structural differences between GDP- and GTP-tubulin in situ. The change of tubulin-pitch by GTP-hydrolysis was also found. This is the first demonstration to show structural changes of MTs under physiological conditions.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Modelling X-ray Diffraction From The Myosin Superlattice Of Vertebrate Muscle

Muscular force is generated by molecular interactions between the contractile proteins actin and myosin. The myosin filaments in the sarcomere of vertebrate muscle pack on a triangular array into which the actin filaments are interdigitated. High resolution studies of the actin-myosin interactions are performed by x-ray fiber diffraction analysis of whole muscle fibers. In most vertebrate muscles however, the myosin filaments pack in a so-called “superlattice” arrangement that involves a semi-random distribution of two filament rotations. The unknown effects of this disorder on diffraction by muscle fibres have so far prevented a rigorous analysis of x-ray fiber diffraction patterns in terms of the structure of this complex system. We report a quantitative model of the disorder and its incorporation into calculations of x-ray fiber diffraction patterns from model structures. This allows rapid calculation of the diffraction and does not involve numerical averaging over the disorder. Calculations show that the disorder modulates the Bragg reflections in diffraction patterns and introduces diffuse diffraction. The results of this analysis will allow the effects of the disorder to be included in muscle structure refinement programs, allowing more accurate structure determination from x-ray fiber diffraction data.

2P167 X線繊維回折により捉えた鞭毛紬糸の構造変化(分子モーター,ポスター発表,第45回日本生物物理学会年会)

2P167 X線繊維回折により捉えた鞭毛紬糸の構造変化(分子モーター,ポスター発表,第45回日本生物物理学会年会)

Position and Orientation of Phalloidin in F-Actin Determined by X-Ray Fiber Diffraction Analysis

Knowledge of the phalloidin binding position in F-actin and the relevant understanding of the mechanism of F-actin stabilization would help to define the structural characteristics of the F-actin filament. To determine the position of bound phalloidin experimentally, x-ray fiber diffraction data were obtained from well-oriented sols of F-actin and the phalloidin-F-actin complex. The differences in the layer-line intensity distributions, which were clearly observed even at low resolution (8 Å), produced well-resolved peaks corresponding to interphalloidin vectors in the cylindrically averaged difference-Patterson map, from which the radial binding position was determined to be ∼10 Å from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be ∼84° and 0.5 Å relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH) 7 residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11.

MM3 potential energy surfaces of trisaccharides. II. Carrageenan models containing 3,6-anhydro-D-galactose.

The adiabatic potential energy surfaces (PES) of six trisaccharides-namely 3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp, beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp, and their derivatives sulfated on positions 2 and 4 of the beta-galactose unit-were obtained using the MM3 force field. Each PES was described by a single contour map for which the energy is plotted against the two psi glycosidic angles, given the small variations of the phi glycosidic torsional angle in the low-energy regions of disaccharide maps. In five of the six examples, the surfaces are those expected from the maps of the disaccharidic repeating units of carrageenans, with less important factors altering the additive effect of both linkages. However, when a sulfate group is present on C2 of a beta-galactose reducing end, a new low-energy minimum in a different region is produced, originated in a hydrogen bond between the first and third monosaccharidic moieties of the trisaccharide. The flexibility of the beta-linkages is nearly identical to that in their disaccharide counterparts, while that of the alpha-linkages is slightly reduced, independent of their presence closer or further away from the reducing end. A fair agreement is observed between the x-ray fiber diffraction analysis for a kappa-carrageenan double helix and the surfaces obtained for the trisaccharide analogs of that polymer.

X-ray fibre diffraction analysis of assemblies formed by prion-related peptides: Polymorphism of the heterodimer interface between PrPCand PrPSc

The molecular mechanism in prion diseases involves structural alteration of the non-infectious cellular isoform (PrPC) to the infectious, scrapie isoform (PrPSc). The structural transition is thought to involve a binding interface between the two isoforms in their N-terminal domains. Because prions and prion-related peptides can form amyloid-like fibrillar assemblies, we have used X-ray fibre diffraction to study the structure of the binding interface in synthetic peptides that have sequence similarity with the prion protein (PrP). Our previous studies show that the alanine-rich peptides PrP106-122 and PrP109-122 form slab-like structures that are stacked as one-dimensional lattices having cumulative disorder. The unit cell is a fourstranded β-sheet with the chains directed along the stacking direction. Neighboring β-sheets are quarterstaggered (like in β silk). Electron density projections indicate that the peptides form a reverse turn with the larger residues in the N-terminal domain distinguishable from the alanine-rich C-terminal domain. This reverse turn accounts for the 33 A-length of β chains (or slab thickness) as measured from the low-angle scattering. In this report, we considered three different inter-molecular packings for the reverse turn molecules: i.e., anti-parallel, parallel, and staggered. The staggered arrangement gave the best agreement between the observed and calculated X-ray intensities. From the molecular model we suggest that the autocatalytic replication of prions may involve hydrogen-bonding between intermolecular antiparallel β chains of the alanine-rich domains at the binding interface between PrPC and PrPSc.

Effect of calcium ions on the organization of iota-carrageenan helices: an X-ray investigation

X-ray fiber diffraction analysis confirms that calcium ι-carrageenan forms a threefold, right-handed, half-staggered, parallel, double helix of pitch 26.42 Å stabilized by interchain hydrogen bonds. According to the detailed structural results, three helices are packed in a trigonal unit cell ( a=23.61 and c=13.21 Å). Strong interactions between the sulfate groups of neighboring helices, mediated by calcium ions and water molecules, are responsible for stabilizing the three-dimensional structure.

X-ray structure analysis of the sodium salt of beijeran

The three-dimensional structure of the sodium salt of beijeran has been determined by X-ray fiber diffraction analysis. The acidic polysaccharide forms an extended twofold helix. Two chains are nestled tightly in a monoclinic unit cell of dimensions a=12.72, b=11.41, c (fiber axis)=24.62 Å and γ=123.7° in an antiparallel fashion. In the crystalline lattice, helices are stacked tightly to form a thick sheet along the vertical plane passing through the short diagonal of the basal net. Adjacent sheets associate via a network of sodium ions and water molecules embedded between them. The morphology of sodium beijeran in the solid state is consistent with its observed rheological properties.

Thermally reversible acid-induced gelation of low-methoxy pectin

Gelation of low-methoxy pectin (DE 31.1) on cooling under acidic conditions in the absence of Ca 2+ has been investigated by rheological measurements under low-amplitude oscillatory shear. The mechanical spectra obtained after 60 min at 5°C showed a progressive increase in solid-like response (increasing G′; decreasing tan δ; increasing frequency-dependence of η ∗) as the pH was reduced from 4.0 to 1.6, with formation of a critically crosslinked network at ∼pH 3.0 (for a polymer concentration of 3.0 wt%). By extrapolation from X-ray fibre diffraction analysis of pectic acid, it is suggested that crosslinking occurs by association of three-fold helices. At pH values between ∼3.5 and ∼2.5 there is no detectable thermal hysteresis between the sol–gel transition on cooling and gel–sol transition on heating, and both are accompanied by a sigmoidal change in optical rotation (attributed to formation and melting of three-fold order). Substantial hysteresis is, however, observed at lower and higher pH, and is attributed to extensive aggregation as electrostatic repulsion is suppressed (below ∼pH 2.5) and slow formation of intermolecular hydrogen bonds by protonated carboxyl groups (above ∼pH 3.5), respectively. The transition enthalpy from DSC heating scans has a maximum value of ΔH≈11 J/g at ∼pH 3.0, but decreases sharply at lower and higher pH, with accompanying loss of a detectable transition in optical rotation. It is suggested that the chain conformation in solution at low pH is predominantly three-fold with, therefore, little conformational change on adoption of the ordered, intermolecular structure, whereas at high pH the solution conformation is predominantly two-fold, with only limited conversion to the three-fold (acid) form on cooling.

Syntheses, Characterizations, and Structures of a New Series of Aliphatic−Aromatic Polyesters. 1. The Poly(tetramethylene terephthalate dicarboxylates)

A new family of polyesters, the poly(tetramethylene terephthalate dicarboxylates) of formula [−O−CO−C6H4−CO−O−(CH2)x−O−CO−(CH2)y−CO−O−(CH2)x]n, was synthesized and characterized by 1H and 13C NMR in CDCl3 solution, infrared spectroscopy, molecular weight distribution, and X-ray fiber diffraction analysis. The 1H and 13C NMR spectroscopy and the molecular weight distribution confirm the homogeneous alternating distribution of the two kinds of diacids (aromatic and aliphatic). The crystal structures of the adipate and the succinate were determined by interpretation of their X-ray diffraction patterns. A dynamic infrared spectroscopic study confirmed the existence of a nongauche−nontrans torsion angle in the glycol segments, while the diacid moieties were in a nearly all-trans conformation.

Model bias in Bayesian image reconstruction from x-ray fiber diffraction data

An image-reconstruction problem in x-ray fiber diffraction analysis (which is used to determine the atomic structure of biopolymers) is considered. The problem is to reconstruct an image (the electron density function) given data that are squared sums of the Fourier coefficients of the image as well as partial information (the model) on the image. A Bayesian estimation approach based on a prior for the missing part of the image is considered. Current (heuristic) approaches to this problem correspond to certain maximum a posteriori estimates. These estimates exhibit bias toward the model, and current methods to reduce the bias are based on scaling of the Fourier coefficients. A new procedure to remove bias, based on orthogonalization, is derived and shown by simulations to be superior to scaling. Bias and unbiasing are compared for the different maximum a posteriori estimates, for different amounts of missing information. These results are also compared with a new minimum mean-square-error estimate for this problem that has the form of weighted maximum a posteriori Fourier coefficients. The minimum mean-square-error estimate is free from bias and gives results superior to the unbiased maximum a posteriori estimates.

Bayesian image reconstruction from partial image and aliased spectral intensity data

An image reconstruction problem motivated by X-ray fiber diffraction analysis is considered. The experimental data are sums of the squares of the amplitudes of particular sets of Fourier coefficients of the electron density, and a part of the electron density is known. The image reconstruction problem is to estimate the unknown part of the electron density, the "image." A Bayesian approach is taken in which a prior model for the image is based on the fact that it consists of atoms, i.e., the unknown electron density consists of separated, sharp peaks. Currently used heuristic methods are shown to correspond to certain maximum a posteriori estimates of the Fourier coefficients. An analytical solution for the Bayesian minimum mean-square-error estimate is derived. Simulations show that the minimum mean-square-error estimate gives good results, even when there is considerable data loss, and out-performs the maximum a posteriori estimates.

Crystal Structure of Valonia Cellulose Iβ

The crystal structure of Valonia cellulose Iβ is determined by X-ray fiber diffraction analysis. A careful reanalysis of two existing X-ray diffraction data sets for Valonia cellulose I leads to a consistent, definitive structure of the β-phase. The results resolve ambiguities in existing X-ray analyses between “parallel up” and “parallel down” packing of the cellulose sheets in native Valonia cellulose. The molecular and sheet structures are essentially identical to those previously determined, but these results define precisely the packing of the sheets. The sheet packing is discussed in terms of the energy and density of the crystal structure. The structure of the β-phase has specific implications for the packing of the α-phase of Valonia cellulose I.