Fiber Diffraction Data Research Articles

X-ray scattering and molecular dynamics simulations reveal the secondary structure of κ-carrageenan in the solution state

The solution state structure of κ-carrageenan is typically described as a ‘random coil’, to indicate a lack of defined secondary structure elements. From this starting point the assignment of an optical-rotation-detected change that follows the introduction of particular ions to such solutions as a ‘coil-to-helix transition’ seems unambiguous, and thus the canonical description of this important biopolymer's gelling behaviour was born. However, the notion that κ-carrageenan exists in solution as a random coil, devoid of secondary structure, has been questioned a number of times previously in the literature, particularly by the molecular modelling and NMR communities. Regrettably, there has been little desire to-date to address these largely overlooked studies or consider their implications for the nature of the so-called ‘coil-to-helix transition’. Despite evidence to the contrary, the random-coil-paradigm has prevailed. Here, new data from synchrotron-enabled solution-state x-ray scattering experiments, combined with state-of-the-art atomistic molecular dynamics simulations, are used to show that the solution-state structure of κ-carrageenan in fact retains many of the helical motifs present in the solid-state, as inferred from fibre diffraction data. Furthermore, no evidence is found to suggest that single chains undergo any uni-molecular conformational transition upon the addition of ions. These findings once again challenge the paradigm that κ-carrageenan exists as a ‘random coil’ in the solution state, and thereby question the long held assumption that a uni-molecular ‘coil-to-helix transition’ precedes the dimerization of helices.

Cooperative Induction of Ordered Peptide and Fatty Acid Aggregates

Interactions between biological membranes and disease-associated amyloids are well documented, and their prevalence suggests that an inherent affinity exists between these molecular assemblies. Our interest in the molecular origins of life have led us to investigate the nature of such interactions in the context of their molecular predecessors (i.e., vesicle-forming amphiphiles and small peptides). Under certain conditions, amyloidogenic peptides or fatty acids are each able to form ordered structures on their own; however, we report here on their cooperative assembly into novel, to our knowledge, highly ordered structures. We first examined an amyloidogenic eight-residue peptide, which forms amyloids at pH 11, yet because of its positive electrostatic character remains soluble at a neutral pH. In mixtures with simple fatty acids, this peptide is also able to form novel, to our knowledge, coaggregates at a neutral pH whose structures are sensitive to both the fatty acid concentration and identity. Below the critical vesicle concentration, the mixtures of fatty acid and peptide yield a flocculent precipitate with an underlying β-structure. Above the critical vesicle concentration, the mixtures yield a translucent precipitate that consists of tube-like structures. Small-angle x-ray scattering and fiber diffraction data were used to model their structures as hollow-core two-shell cylinders in which the inner shell is a bilayer of fatty acid and the outer shell alternates between amyloid and bilayers of fatty acid. The further analysis of decanoic acid with a panel of 13 other basic amyloidogenic peptides confirmed the general nature of the observed interactions. The cooperativity within this heterogeneous system is attributed to the structurally repetitive natures of the fatty acid bilayer and the cross-β-sheet motif, providing compatible scaffolds for attractive electrostatic interactions. We show these interactions to be mutually beneficial, expanding the phase space of both peptides and fatty acids while providing a simple yet robust physical connection between two distinct entities relevant for life.

Polymorphism in disease-related apolipoprotein C-II amyloid fibrils: a structural model for rod-like fibrils.

Human apolipoprotein (apo) C-II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid-free apoC-II readily self-assembles into twisted-ribbon amyloid fibrils but forms straight, rod-like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod-like fibril formation compared to their effects on twisted-ribbon fibril formation. For instance, the double mutant, K30D-D69K apoC-II, readily formed twisted-ribbon fibrils, while the rate of rod-like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod-like apoC-II fibrils, using hydrogen-deuterium exchange and NMR analysis showed exchange protection consistent with a core cross-β structure comprising the C-terminal 58-76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross-β structure. X-ray fibre diffraction data for aligned rod-like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted-ribbon fibrils and are predicted for a model involving two cross-β fibrils in an off-set antiparallel structure with four apoC-II units per rise of the β-sheet. This model is consistent with the mutational effects on rod-like apoC-II fibril formation. The lipid-dependent polymorphisms exhibited by apoC-II fibrils could determine the properties of apoC-II in renal amyloid deposits and their potential role in the development of cardiovascular disease.

X-ray crystal structure of anhydrous chitosan at atomic resolution.

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X-ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P21 21 21 ] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361-368, 2016.

A computational model for predicting experimental RNA nearest-neighbor free energy rankings: Inosine·uridine pairs

A computational model for predicting RNA nearest neighbor free energy rankings has been expanded to include the nonstandard nucleotide inosine. The model uses average fiber diffraction data and molecular dynamic simulations to generate input geometries for Quantum mechanic calculations. This resulted in calculated intrastrand stacking, interstrand stacking, and hydrogen bonding energies that were combined to give total binding energies. Total binding energies for RNA dimer duplexes containing inosine were ranked and compared to experimentally determined free energy rankings for RNA duplexes containing inosine. Statistical analysis showed significant agreement between the computationally determined rankings and the experimentally determined rankings.

Automated determination of fibrillar structures by simultaneous model building and fiber diffraction refinement.

For highly oriented fibrillar molecules, three-dimensional structures can often be determined from X-ray fiber diffraction data. However, because of limited information content, structure determination and validation can be challenging. We demonstrate that automated structure determination of protein fibers can be achieved by guiding the building of macromolecular models with fiber diffraction data. We illustrate the power of our approach by determining the structures of six bacteriophage viruses de novo using fiber diffraction data alone and together with solid-state NMR data. Furthermore, we demonstrate the feasibility of molecular replacement from monomeric and fibrillar templates by solving the structure of a plant virus using homology modeling and protein-protein docking. The generated models explain the experimental data to the same degree as deposited reference structures but with improved structural quality. We also developed a cross-validation method for model selection. The results highlight the power of fiber diffraction data as structural constraints.

Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218–289)

The fungal prion-forming domain HET-s(218-289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung β-solenoid structure. Under acidic conditions, HET-s(218-289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218-289) fibrils formed under acidic conditions have a stacked β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.

Insights into the Molecular Architecture of a Peptide Nanotube Using FTIR and Solid‐State NMR Spectroscopic Measurements on an Aligned Sample

Queuing up: Molecular orientation within macroscopically aligned nanotubes of the peptide AAAAAAK can be studied by solid-state NMR and IR spectroscopy. Line shape analysis of the NMR spectra indicates that the peptide N-H bonds are tilted 65-70° relative to the nanotube long axis. Re-evaluation of earlier X-ray fiber diffraction data suggests that the peptide molecules are hydrogen-bonded in a helical arrangement along the nanotube axis.

The Structure of Cross‐β Tapes and Tubes Formed by an Octapeptide, αSβ1

Elaborate morphology: The αSβ1 peptide, a fragment of α-synuclein, assembles into flat tapes consisting of a peptide bilayer, which can be modeled based on the cross-β structure found in amyloid proteins. The tapes are stabilized by hydrogen bonding, whilst the amphiphilic nature of the peptide results in the thin bilayer structure. To further stabilize the structure, these tapes may twist to form helical tapes, which subsequently close into nanotubes.

Water in Crystalline Fibers of Dihydrate β-Chitin Results in Unexpected Absence of Intramolecular Hydrogen Bonding

The complete crystal structure (including hydrogen) of dihydrate β-chitin, a homopolymer of N-acetylglucosamine hydrate, was determined using high-resolution X-ray and neutron fiber diffraction data collected from bathophilous tubeworm Lamellibrachia satsuma. Two water molecules per N-acetylglucosamine residue are clearly localized in the structure and these participate in most of the hydrogen bonds. The conformation of the labile acetamide groups and hydroxymethyl groups are similar to those found in anhydrous β-chitin, but more relaxed. Unexpectedly, the intrachain O3-H…O5 hydrogen bond typically observed for crystalline β,1–4 glycans is absent, providing important insights into its relative importance and its relationship to solvation.

Crystal structure of (Gly‐Pro‐Hyp)9: Implications for the collagen molecular model

Collagens have long been believed to adopt a triple-stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X-ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single-crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly-Pro-Hyp)(9) peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly-Pro-Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly-Pro-Hyp)(9) peptide adopts a triple-stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation.

Elucidating the Mechanism of Collagenolysis in the Native Collagen Fibril

Collagen is an important structural biomolecule that is abundant in all animals. Proper degradation and remodeling of collagen is crucial to an organism's survival; moreover, excessive degradation of collagen is involved in many disease processes such as atherosclerosis, cancer metastasis, and arthritis. While a number of studies have shed light on the mechanism of collagenolysis in solution, the precise mechanism of collagen degradation in the native fiber remains mysterious. In this work, we use molecular dynamics simulations on a model of native collagen fibrils, obtained from recent fiber diffraction data, to understand the mechanism of fibrillar degradation. Our data suggest that the unique collagenase cleavage site in native fibrillar state behaves much like it does in solutions composed of tropocollagen molecules. In particular, in the fibrillar state the region about the collagenase cleavage site can adopt two distinct conformational states - one where the atoms comprising the scissile bond are relatively hidden and another state where the scissile bond is relatively exposed, or vulnerable, to collagenolysis. These data extends our previous work and suggests that conformational flexibility plays an important role in determining the rate of collagenolysis in collagen fibrils.

Computational Model for Predicting Experimental RNA and DNA Nearest-Neighbor Free Energy Rankings

Hydrogen-bonding, intrastrand base-stacking, and interstrand base-stacking energies were calculated for RNA and DNA dimers at the MP2(full)/6-311G** level of theory. Standard A-form RNA and B-form DNA geometries from average fiber diffraction data were employed for all base monomer and dimer geometries, and all dimer binding energies were obtained via single-point calculations. The effects of water solvation were considered using the PCM model. The resulting dimer binding energies were used to calculate the 10 unique RNA and 10 unique DNA computational nearest-neighbor energies, and the ranking of these computational nearest neighbor energies are in excellent agreement with the ranking of the experimental nearest-neighbor free energies. These results dispel the notion that average fiber diffraction geometries are insufficient for calculating RNA and DNA stacking energies.

Notes from abroad on the deciphering of biology's Rosetta Stone.

I worked in Marshall Nirenberg’s group at NIH from September 1962 to September 1964. My admiration and consideration for Marshall and his group’s contribution as recognized recently by the American Chemical Society is enormous. I now draw the parallel between how the Rosetta Stone enabled our culture to learn of the past in Egypt and the decipherment of the Genetic Code, for example, comparing the hieroglyphics and the string of DNA bases. The conformation of DNA was worked out by another hero of mine, Francis Crick, in collaboration with James Watson by glorified inspired guess work. My PhD supervisor, Dan Brown at Cambridge University, determined the chemical structures of DNA and RNA together with Nobel Prize winner Alexander Todd (later Lord Todd). At Dan’s 70th birthday party, Lord Todd averred: ‘‘What we did, Dan was to solve the structure of nucleic acid including DNA. All that Watson and Crick did was to solve the conformation’’. As usual, the great man Todd spoke some basic truths bluntly. Actually Alexander Todd forged the makings of my career. When I was in my last year of Ph.D. studies, Todd acted as my supervisor whilst Dan Brown was on sabbatical leave. Todd asked me what I wanted to do. I replied: ‘biochemistry’. He immediately arranged a Research Associate position at MIT’s Department with Professor J.M. Buchanan, an expert in nucleic acid components’ metabolism. But first let me explain a little here on DNA. Working with Francis Crick was also inspiring as he had ideas about everything, and I was lucky that he spent 6 weeks in my Danish lab as he emigrated to the Salk Institute. When he was here in 1976 in Denmark, some scientists in New Zealand pointed out that the crude X-ray fibre diffraction data on DNA could also be explained by a side-by-side model for the 2 strands. Jim Watson reportedly said: ‘rubbish’. Francis said: ‘it could be’, but he pointed out that he knew the answer because of the work I did with Aaron Klug on solving the 3D structure of tRNA because we showed RNA helices at atomic resolution. If there are helices in RNA then it was pretty certain that helices occurred in DNA. In point of fact, a small piece of double stranded DNA was not solved by X-crystallography until 1978 at Caltech by Dickersen and Drew. I use this story to highlight the importance of Marshall’s work which allowed all biologists and chemists to understand and control the various cellular molecular mechanisms that have had tremendously positive outcomes in molecular medicine for which NIH continues to be famous. The genetic code truly gives us insight into function which the DNA informational strands cannot. How did I reach NIH? Essentially it was due to Marshall’s friends and eminent scientist supporters who acted when Marshall’s discovery in 1961 stimulated a host of competitors. The news of Marshall’s talk at the IUB Congress in Moscow spread quickly. As a later President of the IUB, which included molecular biology in 1991 to become the IUBMB, I wished that I had presided at such a momentous occasion. In the Fall of 1961, Marshall gave a full house seminar at MIT on the synthetic mRNA work showing that polyU made polyPhe. Even Jim Watson who came with people from Harvard did not read his usual paper. The only untoward incident came in the question time when a post doc from NYU (Ochoa’s group) tried to convince the audience that they were also as far ahead in the genetic code as NIH particularly because they discovered the enzyme polynucleotide phosphorylase which was B. F. C. Clark (&) Molecular Biology, Aarhus University, Aarhus, Denmark e-mail: bfcc@mb.au.dk

X-ray Structure of Anhydrous β-Chitin at 1 Å Resolution

The crystal structure of β-chitin, a homopolymer of N-acetylglucosamine, was determined using highly resolved X-ray fiber diffraction data obtained from uniaxially oriented specimen of diatom spines. The labile N-acetyl groups and hydroxymethyl groups were directly located in the Fourier omit maps constructed using the glycosyl backbone as phasing model. Using the 216 independent intensities extending to a resolution of 1 A, we could refine the structure without any restraints on bond angles to obtain a reasonable model with high accuracy. The carbonyl oxygen of acetamide group formed intermolecular hydrogen bonding between the primary hydroxyl and amine groups forming a two-dimensional hydrogen bonding network in a plane perpendicular to the pyranosyl plane. The hydrogen bonding pattern explains why the conformation of the acetamide groups is slightly rotated from standard conformation.

Molecular and structural mapping of collagen fibril interactions

The fibrous collagens form the structural basis of all mammalian connective tissues, including the vasculature, dermis, bones, tendons, cartilage, and those tissues that support organs such as the heart, kindneys, liver, and lungs. The helical structure of collagen has been extensively studied but in addition to its helical character, its molecular packing arrangement (in its aggregated or fibrillar form) and the presence of specific amino acid sequences govern collagen's in vivo functions. Collagen's molecular packing arrangement helps control cellular communication, attachment and movement, and conveys its tissue-specific biomechanical properties. Recent progress in understanding collagen's molecular packing, fibrillar structure, domain organization, and extracellular matrix (ECM) interactions in light of X-ray fiber diffraction data provides significant new insights into how the ECM is organized and functions. In this review, the hierarchy of fibrillar collagen structure is discussed in the context of how this organization affects ECM–“ligand” interactions, with specific attention to collagenolysis, integrins, fibronection, glycoprotein VI receptor (GPVI), and proteoglycans (PG). Understanding the complex structure of collagen and its attached ligands should provide new insights into tissue growth, development, regeneration, and disease.

Consensus structure of Pf1 filamentous bacteriophage from X-ray fibre diffraction and solid-state NMR.

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.

Architecture of the potyviruses

X-ray fiber diffraction data were obtained and helical pitch and symmetry were determined for seven members of the family Potyviridae, including representatives from the genera Potyvirus, Rymovirus, and Tritimovirus. The diffraction patterns are similar, as expected. There are, however, significant variations in the symmetries, as previously found among the flexible potexviruses, but not among the rigid tobamoviruses. Wheat streak mosaic virus, the only member of the genus Tritimovirus examined, displayed the largest deviations in diffraction data and helical parameters from the other viruses in the group.

Molecular Modeling of the Misfolded Insulin Subunit and Amyloid Fibril

Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly α-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using β-solenoid folds, namely, the β-helix and β-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the β-roll subunit model may be more stable than the β-helix subunit model. We also constructed β-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted β-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies.

Three‐dimensional reconstruction of biological macromolecular complexes from in‐lens scanning electron micrographs

Two helical samples: F-actin and the bacteriophage T4 tail sheath were reconstructed in three dimensions from contrast enhanced (rotational shadowing and negatively stained) in-lens cryo-field emission scanning electron micrographs, using the iterative real-space helical reconstruction method. The F-actin--and bacteriophage T4 reconstructions compare favourably to an atomic model refined against fibre diffraction data and a cryo-electron microscopy reconstruction, respectively. These results show that single-particle methods, developed for macromolecules imaged in the transmission electron microscope can be applied to cryo-field emission scanning electron micrographs data with appropriate symmetry.