X-ray Fiber Diffraction Research Articles

Designing Highly Ordered Helical and Nonhelical Porous Crystalline and Disordered Nonhelical Columnar Liquid Crystalline Self-Organizations.

Helical self-organizations are equilibrium structures responsible for the assembly of nonequilibrium and equilibrium living and synthetic systems. Racemic helical columnar systems transform into one-handed systems with the help of enantiomerically rich or pure components. Racemic, enantiomerically rich, and enantiomerically pure helical periodic arrays of columns are analyzed by oriented fiber X-ray diffraction (XRD). With few exceptions, highly ordered helical 3-D organizations as generated from homochiral columns cannot be obtained from achiral, racemic, or enantiomerically rich helical columns. Here, we report an unprecedented class of nonhelical porous ordered, disordered nonhelical columnar liquid crystalline (LC) self-organizations and columnar liquids constructed from AB4 to AB9 isomeric terphenyls by molecular design unwinding of a 3-D helical organization. A library of 16 nonhelical porous ordered, disordered columnar and four liquids was designed by employing as a model a closely related achiral AB4 meta-terphenyl, which self-organizes one of the most perfect synthetic ordered columnar hexagonal helices known. A general molecular mechanism to unwind highly ordered 3-D helices into nonhelical porous columnar ordered LCs and liquids was elaborated to design this transformation, which provided unprecedented nonequilibrium synthetic systems. This methodology is expected to be general for transformation of helical macromolecular and supramolecular organizations into nonhelical crystals, LCs, and liquids.

MuscleX: data analysis software for fiber diffraction patterns from muscle.

MuscleX is an integrated, open-source computer software suite for data reduction of X-ray fiber diffraction patterns from striated muscle and other fibrous systems. It is written in Python and runs on Linux, Microsoft Windows or macOS. Most modules can be run either from a graphical user interface or in a `headless mode' from the command line, suitable for incorporation into beamline control systems. Here, we provide an overview of the general structure of the MuscleX software package and describe the specific features of the individual modules as well as examples of applications.

Monomer sequence segregation in the liquid crystalline phase of thermotropic copolyester amide. In-situ X-ray scattering

In-situ wide-angle X-ray scattering of a sheared thermotropic random copolyester amide revealed monomeric sequence segregation in the liquid crystalline phase. The copolyester amide is based on 60 mol% 2,6-hydroxynaphthoic acid, 20 mol% terephthalic acid and 20 mol% amino phenol (designated N-T-AP). The solid phase exhibits unusual crystallinity and the fiber X-ray diffraction exhibits aperiodic meridional reflections consistent with sequence segregation along the chain. In the liquid crystalline phase at 305 °C polarized optical microscopy showed a “worm” texture, and X-ray scattering showed an amorphous halo denoting no preferred macromolecular orientation. Upon application of shear the X-ray pattern became anisotropic with an increase of intensity on the equatorial axis consistent with the macromolecules being aligned along the flow axis. Strikingly, the X-ray pattern also exhibited a meridional reflection in the melt indicative of spatial monomer register along chains axes. Increasing the shear rate increased the equatorial intensity and reduced the azimuthal spread of the meridional reflection corresponding to an increase of longitudinal monomer register. The monomer register was preserved over a range of temperatures and upon quenching the polymer under shear. Therefore, the unusual crystallinity in solid state would be explained by the existence of monomer sequence segregation already present in the melt.

Atomic Scale Structure of Self-Assembled Lipidated Peptide Nanomaterials.

β-Peptides have great potential as novel biomaterials and therapeutic agents, due to their uniqueability to self-assemble into low dimensional nanostructures,and their resistance to enzymatic degradationin vivo. However, the self-assembly mechanisms of β-peptides, which possess increased flexibility due to the extra backbone methylene groups present within the constituent β-amino acids, are not well understood due to inherent difficulties of observing their bottom-up growth pathway experimentally. A computational approach is presented for the bottom-up modelling of the self-assembledlipidated β3-peptides, from monomers, to oligomers, to supramolecular low-dimensional nanostructures, in all-atom detail. The approach is applied to elucidate the self-assembly mechanisms of recently discovered, distinct structural morphologies of low dimensional nanomaterials, assembled from lipidated β3-peptide monomers. The resultant structures of the nanobelts and the twisted fibrils are stable throughout subsequent unrestrained all-atom molecular dynamics simulations, and these assemblies display good agreement with the structural features obtained from X-ray fiber diffraction and atomic force microscopy data.This is the first reported, fully-atomistic model of a lipidated β3-peptide-based nanomaterial, and the computational approach developed here, in combination with experimental fiber diffraction analysis and atomic force microscopy, will be useful in elucidating the atomic scale structure of self-assembled peptide-based and other supramolecular nanomaterials.

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure With Pulmonary Hypertension.

Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in patients with heart failure with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. We thus sought to characterize RV myocyte contractile depression in HFrEF-PH, identify those components reflected by clinical RV indices, and uncover underlying biophysical mechanisms. Resting, calcium-, and load-dependent mechanics were prospectively studied in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 patients with HFrEF-PH undergoing cardiac transplantation and 9 organ donor controls. Unsupervised machine learning using myocyte mechanical data with the highest variance yielded 2 HFrEF-PH subgroups that in turn mapped to patients with decompensated or compensated clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in decompensated clinical RV function, whereas surprisingly, many other major myocyte contractile measures including peak power and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick filament defects, myofibrillar structure was assessed by x-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in decompensated clinical RV function, but not compensated clinical RV function, as compared with controls. This corresponded to reduced myosin ATP turnover in decompensated clinical RV function myocytes, indicating less myosin in a crossbridge-ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Last, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human heart failure. Although there are many RV myocyte contractile deficits in HFrEF-PH, commonly used clinical indices only detect reduced isometric calcium-stimulated force, which is related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

Amyloid fibril formation, structure and domain swapping of acyl-coenzyme A thioesterase-7.

Acyl-coenzyme A thioesterase (Acot) enzymes are involved in a broad range of essential intracellular roles including cell signalling, lipid metabolism, inflammation and the opening of ion channels. Dysregulation in lipid metabolism has been linked to neuroinflammatory and neurological disorders such as Alzheimer's and Parkinson's diseases. Structurally, Acot enzymes adopt a circularised trimeric arrangement with each monomer containing an N- and a C-terminal hotdog domain. Acot7 spontaneously forms amyloid fibrils in vitro under physiological conditions. The resultant amyloid fibrillar structures were characterised by dye-binding fluorescence assays, far-UV circular dichroism spectroscopy, transmission electron microscopy and X-ray fibre diffraction. Acot7 has an unusual mechanism of aggregation with no lag phase. The initial phase (~ 18 h) of aggregation involves conformational rearrangement within the oligomers to form species of enhanced β-sheet character. The subsequent loss of α-helical structure is accompanied by large-scale amyloid fibril formation. The crystal structure of Acot7 revealed an unexpected arrangement of the two domains within the circularised trimeric structure, which is the basis for a proposed mechanism of amyloid fibril formation involving domain swapping during the initial phase of aggregation. Acot7 formed fibrils in the presence of its substrate arachidonoyl-CoA and its inhibitors and maintained its enzyme activity during fibril assembly. It is proposed that the Acot7 fibrillar form acts as functional amyloid.

Structural insight into the stabilization of microtubules by taxanes.

Paclitaxel (Taxol®) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drug's mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 Å resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the bM loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, the longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is, however, not related to the microtubule stabilization mechanism of taxanes, as all analogs tested expand the microtubule lattice, despite the fact that one of them, Baccatin III, is biochemically inactive. In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.

Titin function in the active sarcomere studied in frog muscle fibers upon inhibition of myosin motors by para-nitro-blebbistatin.

Titin spans the whole half-sarcomere, through the I-band connecting the Z-line to the myosin filament tip, and through the A-band, associated to myosin filament, up to M-line. Titin in the I-band transmits the stress also without attached myosin motors and is responsible for the passive force developed when a skeletal muscle fibre is stretched at rest. However, the passive stiffness in the range of physiological sarcomere lengths (SL, <2.7 µm) is too low to account for a putative role of I-band titin as mechanical link able to sustain mechanosensing in myosin filaments and prevent development of sarcomere inhomogeneity during contraction. Here we define titin mechanics during tetanic stimulation of intact frog muscle fibers using 20 μM para-nitro-blebbistatin (which fully inhibits myosin ATPase in vitro) to suppress the myosin-based mechanical responses. Small-angle X-ray fiber diffraction at the European Synchrotron ESRF demonstrates that the resting structure of thick filament is fully preserved upon tetanic stimulation. Under these conditions we use stepwise changes in force (10-50 pN per half-thick filament, htf) to elicit the lengthening transient that defines titin dynamic stiffness in situ at 2.3–3.0 µm SL. We show that, upon tetanic stimulation titin switches from the OFF-state characterised by a large, SL-dependent, extensibility, to the ON-state in which it opposes stretching with an effective stiffness one-two order of magnitude larger (∼3 pN·nm−1·htf−1) and independent of SL. The results demonstrate that, in the active sarcomere working at physiological lengths, I-band titin is an efficient mechanical link between the Z-line and the myosin filament tip, a condition for playing a role in mechanosensing-based myosin filament activation and for opposing to development of SL inhomogeneity during contraction. Supported by ECRF, University of Florence and MUR (Italy), ESRF (France) and EJP-RD.

Interpreting equatorial X-ray diffraction patterns from striated muscle with multiscale modeling.

Small-angle X-ray diffraction is the tool of choice to obtain nm-scale structural information from striated muscle under near-physiological conditions. A critical barrier to progress has been the lack of generally applicable computational tools for modeling these X-ray diffraction patterns, limiting our ability to extract all the structural information potentially available. We addressed this need by extending the spatially explicit simulation platform MUSICO to predict equatorial fiber X-ray diffraction patterns from skeletal muscle. Electron densities for thin filaments with bound crossbridges were calculated using PDB structures of myosin S1, actin, tropomyosin, troponin and analogous nebulin structure derived from tropomyosin PDB. Thick filament backbone densities were constructed based on the Squire 1973 model incorporating “ribbon motifs” from a high-resolution cryo-EM structure of the Lethocerus thick filament. Titin and myosin S2 regions were constructed as cylindrical rings of density shells. Myosin heads in an ordered “parked state” were positioned in a shell close to the thick filament backbone with any disordered myosin heads distributed in a shell extending out to actin. X-ray diffraction patterns were calculated from sub-system motifs consisting of 1 thick filament and two thin filaments by evaluating the Fourier transform of this object at the reciprocal hexagonal lattice positions. Intensities were averaged over the 500 thick filaments and 1000 thin filaments used in simulation. Using this approach, we found that we could recapitulate 7 independent experimental intensities (out to the 4,0 reflection) from relaxed rat soleus and EDL muscle by varying only the proportion of heads in the parked state and a temperature factor type disorder term. Fits to contracting data could be achieved by adjusting only the temperature factor term and the number of force-producing crossbridges predicted by MUSICO to match the force levels observed experimentally.

Cardiac myosin filaments are directly regulated by calcium.

Classically, striated muscle contraction is initiated by calcium (Ca2+)-dependent structural changes in regulatory proteins on actin-containing thin filaments, which allow the binding of myosin motors to generate force. Additionally, dynamic switching between resting off and active on myosin states has been shown to regulate muscle contractility, a recently validated mechanism by novel myosin-targeted therapeutics. The molecular nature of this switching, however, is not understood. Here, using a combination of small-angle x-ray fiber diffraction and biochemical assays with reconstituted systems, we show that cardiac thick filaments are directly Ca2+-regulated. We find that Ca2+ induces a structural transition of myosin heads from ordered off states close to the thick filament to disordered on states closer to the thin filaments. Biochemical assays show a Ca2+-induced transition from an inactive super-relaxed (SRX) state(s) to an active disordered-relaxed (DRX) state(s) in synthetic thick filaments. We show that these transitions are an intrinsic property of cardiac myosin only when assembled into thick filaments and provide a fresh perspective on nature's two orthogonal mechanisms to regulate muscle contraction through the thin and the thick filaments.

Amyloid fibril formation by αS1- and β-casein implies that fibril formation is a general property of casein proteins

Caseins are a diverse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, αS2- and κ-casein, is their propensity in vitro to form amyloid fibrils, the highly ordered protein aggregates associated with many age-related, including neurological, diseases. In this study, we explored whether amyloid fibril-forming propensity is a general feature of casein proteins by examining the other cow caseins (αS1 and β) as well as β-caseins from camel and goat. Small-angle X-ray scattering measurements indicated that cow αS1- and β-casein formed large spherical aggregates at neutral pH and 20°C. Upon incubation at 65°C, αS1- and β-casein underwent conversion to amyloid fibrils over the course of ten days, as shown by thioflavin T binding, transmission electron microscopy, and X-ray fibre diffraction. At the lower temperature of 37°C where fibril formation was more limited, camel β-casein exhibited a greater fibril-forming propensity than its cow or goat orthologues. Limited proteolysis of cow and camel β-casein fibrils and analysis by mass spectrometry indicated a common amyloidogenic sequence in the proline, glutamine-rich, C-terminal region of β-casein. These findings highlight the persistence of amyloidogenic sequences within caseins, which likely contribute to their functional, heterotypic self-assembly; in all mammalian milks, at least two caseins coalesce to form casein micelles, implying that caseins diversified partly to avoid dysfunctional amyloid fibril formation.

X-ray fiber diffraction reveals ultrastructural location and interactions of the immunoglobulin receptor binding sequence within fibrillar type I collagen, with implications for fibrosis and autoimmune conditions

X-ray fiber diffraction reveals ultrastructural location and interactions of the immunoglobulin receptor binding sequence within fibrillar type I collagen, with implications for fibrosis and autoimmune conditions

X-ray fiber diffraction to elucidate tissue transition and changes to molecular packing in relation to damage

X-ray fiber diffraction to elucidate tissue transition and changes to molecular packing in relation to damage

Molecular parameters including fluorination program order during hierarchical helical self-organization of self-assembling dendrons

Primary structure endows tertiary structure while tertiary structure determines function. This fundamental principle is responsible for the generation of high order and precise functions in both biological assemblies including giant and mega proteins and nucleic acids as well as in metals generated from only few atoms. However, this size-independent ability to generate high order and very precise functions is not yet accessible in non-biological assemblies that use the same principles for their hierarchical self-organization. Here we report a study investigating the role of the molecular parameters on the hierarchical self-organizations of helical columnar assemblies produced from the column-forming self-assembling dendron (4-3,4-3,5)-X with a linear or branched hydrogenated or linear semifluorinated alkyl groups while apex is -CO2CH3 or -CH2OH. A combination of differential scanning calorimetry, small and wide-angle powder and oriented fiber X-ray diffraction experiments together with helical diffraction theory, 2-D and 3-D electron density maps were employed in these structural analysis experiments. They demonstrated that H-bonding at the apex of the self-assembling dendron enhanced the order of the helical columns by comparison with the order of supramolecular helical columns based on dendrons containing an ester group at their apex. This was the case for both linear and branched alkyl groups at the periphery of the self-assembling dendron. Unexpectedly, self-assembling dendrons containing semifluorinated alkyl groups on their periphery and H-bonding groups at their apex increase even more the order of their supramolecular perfluorinated porous helical column. In all cases helix is mediated by the aromatic part of the dendron but hierarchically influenced both by the apex and peripheral groups. A combination of branched alkyl groups with H-bonding at the apex produced a new mechanism to transform helical columns assembled from the column-forming (4-3,4-3,5)-X self-assembling dendron into spherical assemblies, organized from conical dendron conformers, forming an A15 Frank-Kasper phase.

Disk-shaped cellulose fibers from red algae, Eucheuma cottonii and its use for high oxygen barrier

We could prepare disk-shaped fibers without particular mechanical treatments from Eucheuma cottonii, the commonly used red algae for obtaining carrageenan. After carrageenan extraction from cottonii, the residues were bleached using chlorine dioxide and hydrogen peroxide. The morphology of the bleached fiber was disk-shaped one with a very thin fiber wall thickness of less than 100 nm and a diameter of approximately 100 μm. The sugar analysis and X-ray diffraction of the bleached fibers showed that they consisted of mostly glucose and had the same pattern as cellulose I with more than 50% crystalline structure, respectively. Compared to one-dimensional cellulose micro- or nanofibrils, which exhibits slow drainage and possess intolerably high drying energy, these two-dimensional disk-shaped fibers, when formed a layer in water medium, exhibit fast drainage and low drying energy. The formed sheet resulted in excellent transparency and high oxygen barrier property. Therefore, by using these disk-shaped, thin fibers from cottonii, we expect that the biodegradable and transparent oxygen barrier layer can be produced at a paper machine, which is, if possible, extremely difficult in the case of cellulose micro- or nanofibrils due to their slow drainage and high drying energy.

Small Angle X-ray Diffraction as a Tool for Structural Characterization of Muscle Disease.

Small angle X-ray fiber diffraction is the method of choice for obtaining molecular level structural information from striated muscle fibers under hydrated physiological conditions. For many decades this technique had been used primarily for investigating basic biophysical questions regarding muscle contraction and regulation and its use confined to a relatively small group of expert practitioners. Over the last 20 years, however, X-ray diffraction has emerged as an important tool for investigating the structural consequences of cardiac and skeletal myopathies. In this review we show how simple and straightforward measurements, accessible to non-experts, can be used to extract biophysical parameters that can help explain and characterize the physiology and pathology of a given experimental system. We provide a comprehensive guide to the range of the kinds of measurements that can be made and illustrate how they have been used to provide insights into the structural basis of pathology in a comprehensive review of the literature. We also show how these kinds of measurements can inform current controversies and indicate some future directions.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Influence of Temperature on the Properties of Cellulose Iβ based on Molecular Dynamics Simulations

Natural plants, such as cotton and linen, are rich in cellulose Iβ. The properties of cellulose Iβ under different temperatures was studied using molecular dynamics simulations. Firstly, the crystal of cellulose Iβ was built. To verify the model, the X-ray fibre diffraction and thermal expansion coefficients were calculated, which were found to agree with experimental results. Then the Mulliken population of the bonds were computed and the movement of the centre chain and hydrogen bonds studied over the range 300-550 K using a PCFF force field. The results of the Mulliken population reveal the three steps of pyrolysis. The higher the temperature is, the more intensely the movement of the centre chain is. However, the impact of temperature on the movement of the centre chain is not obvious. From 300 K to 550 K, the total number of hydrogen bonds decreased by only 20%. Moreocer, the rupture of intrachain hydrogen bonds and the formation of interchain hydrogen bonds at 400 K ~ 450 K temperature occurred.

Arabidopsis thaliana Plant Natriuretic Peptide Active Domain Forms Amyloid-like Fibrils in a pH-Dependent Manner.

Plant natriuretic peptides (PNPs) are hormones that have been extracted from many different species, with the Arabidopsis thaliana PNP (AtPNP-A) being the most studied among them. AtPNP-A is a signaling molecule that consists of 130 residues and is secreted into the apoplast, under conditions of biotic or abiotic stress. AtPNP-A has distant sequence homology with human ANP, a protein that forms amyloid fibrils in vivo. In this work, we investigated the amyloidogenic properties of a 34-residue-long peptide, located within the AtPNP-A sequence, in three different pH conditions, using transmission electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, Congo red and Thioflavin T staining assays. We also utilize bioinformatics tools to study its association with known plant amyloidogenic proteins and other A. thaliana proteins. Our results reveal a new case of a pH-dependent amyloid forming peptide in A. thaliana, with a potential functional role.

The Disease Associated Tau35 Fragment has an Increased Propensity to Aggregate Compared to Full-Length Tau.

Tau35 is a truncated form of tau found in human brain in a subset of tauopathies. Tau35 expression in mice recapitulates key features of human disease, including progressive increase in tau phosphorylation, along with cognitive and motor dysfunction. The appearance of aggregated tau suggests that Tau35 may have structural properties distinct from those of other tau species that could account for its pathological role in disease. To address this hypothesis, we performed a structural characterization of monomeric and aggregated Tau35 and compared the results to those of two longer isoforms, 2N3R and 2N4R tau. We used small angle X-ray scattering to show that Tau35, 2N3R and 2N4R tau all behave as disordered monomeric species but Tau35 exhibits higher rigidity. In the presence of the poly-anion heparin, Tau35 increases thioflavin T fluorescence significantly faster and to a greater extent than full-length tau, demonstrating a higher propensity to aggregate. By using atomic force microscopy, circular dichroism, transmission electron microscopy and X-ray fiber diffraction, we provide evidence that Tau35 aggregation is mechanistically and morphologically similar to previously reported tau fibrils but they are more densely packed. These data increase our understanding of the aggregation inducing properties of clinically relevant tau fragments and their potentially damaging role in the pathogenesis of human tauopathies.