Small-Angle X-Ray Scattering Reveals Compact Domain-Domain Interactions in the N-Terminal Region of Filamin C

Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the N-terminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11–12 and 14–15, which are thus new candidate sites for mechanical regulation.     

Wide-Angle X-Ray Solution Scattering for Protein-Ligand Binding: Multivariate Curve Resolution with Bayesian Confidence Intervals

A new way to use wide-angle x-ray solution scattering to study protein-ligand binding is presented. First, scattering patterns are measured at different protein and ligand concentrations. Multivariate curve resolution based on singular value decomposition and global analysis is applied to estimate the binding affinities and reference patterns (i.e., the scattering patterns of individual components). As validated by simulation, Bayesian confidence intervals provide accurate uncertainty estimates for the binding free energies and reference patterns. Experimental results from several protein-ligand systems demonstrate the feasibility of the approach, which promises to expand the role of wide-angle x-ray scattering as a quantitative biophysical tool.     

Anomalous Small Angle X-Ray Scattering Simulations: Proof of Concept for Distance Measurements for Nanoparticle-Labelled Biomacromolecules in Solution

Anomalous small angle X-ray scattering can in principle be used to determine distances between metal label species on biological molecules. Previous experimental studies in the past were unable to distinguish the label-label scattering contribution from that of the molecule, because of the use of atomic labels; these labels contribute only a small proportion of the total scattering signal. However, with the development of nanocrystal labels (of 50–100 atoms) there is the possibility for a renewed attempt at applying anomalous small angle X-ray scattering for distance measurement. This is because the contribution to the scattered signal is necessarily considerably stronger than for atomic labels. Here we demonstrate through simulations, the feasibility of the technique to determine the end-to-end distances of labelled nucleic acid molecules as well as other internal distances mimicking a labelled DNA binding protein if the labels are dissimilar metal nanocrystals. Of crucial importance is the ratio of mass of the nanocrystals to that of the labelled macromolecule, as well as the level of statistical errors in the scattering intensity measurements. The mathematics behind the distance determination process is presented, along with a fitting routine than incorporates maximum entropy regularisation.     

SASCUBE: An Updated Method of Cubes for Calculation of the Intensity of X-Ray Scattering by Biopolymers in Solution

Biophysics - Abstract—This work describes an updated method of cubes, which allows calculation of the SAS curves for biopolymers in solution on the basis of the coordinates of their atoms...     

Resonant X-Ray Scattering and Absorption for the Global and Local Structures of Cu-modified Metallothioneins in Solution

With Cd and Zn metal ions removed from the native rabbit-liver metallothionein upon unfolding, Cu-modified metallothioneins (Cu-MTs) were obtained during refolding in solutions containing Cu^I or Cu^II ions. X-ray absorption near-edge spectroscopic results confirm the respectively assigned oxidation states of the copper ions in Cu^I-MT and Cu^II-MT. Global and local structures of the Cu-MTs were subsequently characterized by anomalous small-angle x-ray scattering (ASAXS) and extended x-ray absorption fine structure. Energy-dependent ASAXS results indicate that the morphology of Cu^II-MT resembles that of the native MT, whereas Cu^I-MT forms oligomers with a higher copper content. Both dummy-residue simulation and model-shape fitting of the ASAXS data reveal consistently rodlike morphology for Cu^II-MT. Clearly identified Cu-S, Cu-O, and Cu-Cu contributions in the extended x-ray absorption fine structure analysis indicate that both Cu^I and Cu^II ions are bonded with O and S atoms of nearby amino acids in a four-coordination environment, forming metal clusters smaller than metal thiolate clusters in the native MT. It is demonstrated that a combination of resonant x-ray scattering and x-ray absorption can be particularly useful in revealing complementary global and local structures of metalloproteins due to the atom specific characteristics of the two techniques.     

The Structure of High Molecular Weight Ribonucleic Acid in Solution: A Small-Angle X-Ray Scattering Study

Small-angle x-ray scattering studies on an absolute scale have been carried out on isotropic solutions of high molecular weight RNA obtained from ascites tumor cells, E. coli, and yeast. It was found that in all three cases the RNA is composed of short rigid rods (50 to 150Å in length) joined by small flexible regions. The rods account for almost the entire structure (at least 90 per cent); their radius of gyration about the axis and their mass per unit length are similar to those of DNA, suggesting a double-stranded helical structure. The rods are joined in an array forming the compact RNA molecule. On thermal degradation, the molecular superstructure disappears while the rods persist.     

Molecular Evidence for the Last Survivor of an Ancient Kangaroo Lineage

The Australasian marsupial family Macropodidae includes potoroos and bettongs (Potoroinae) as well as larger kangaroos, wallabies, and pademelons (Macropodinae). Perhaps the most enigmatic macropodid is the banded hare wallaby, Lagostrophus fasciatus, a taxon listed as vulnerable by the IUCN. Lagostrophus had traditionally been grouped as a sister-taxon to hare wallabies (Lagorchestes), in a clade with hypsodont macropodines, or intercalated in some other fashion within Macropodinae. Flannery (1983, 1989) proposed a radically different hypothesis wherein Lagostrophus is outside of Macropodinae and is more closely related to extinct sthenurine (short-faced) kangaroos. Given this controversy, we addressed the phylogenetic placement of the banded hare wallaby using molecular sequences for three mitochondrial genes (12S rRNA, valine tRNA, 16S rRNA) and one nuclear gene (protamine P1). Diverse phylogenetic methods all provided robust support for a macropodine clade that excludes the banded hare wallaby. The split between macropodines and the banded hare wallaby was estimated at approximately 20 million years ago (mya) using the Thorne/Kishino relaxed molecular clock method. Whereas our molecular results neither corroborate nor refute the sthenurine hypothesis, since all short-faced kangaroos and their immediate ancestors are extinct, the overriding implication of molecular phylogenetic analyses is manifest: the banded hare wallaby is the only living relict of an ancient kangaroo lineage. Regardless of its precise relationships, special efforts should be directed at conserving this unique and endangered taxon, which has not been recorded from mainland Australia since 1906 and is now restricted to two tiny islands off the coast of Western Australia.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1023/A:1022697300092     

An X-Ray Scattering Study into the Structural Basis of Corneal Refractive Function in an Avian Model

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3–4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.     

An X-Ray Scattering Study into the Structural Basis of Corneal Refractive Function in an Avian Model

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3–4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.     

Beyond the Whole-Genome Duplication: Phylogenetic Evidence for an Ancient Interspecies Hybridization in the Baker's Yeast Lineage

Whole-genome duplications have shaped the genomes of several vertebrate, plant, and fungal lineages. Earlier studies have focused on establishing when these events occurred and on elucidating their functional and evolutionary consequences, but we still lack sufficient understanding of how genome duplications first originated. We used phylogenomics to study the ancient genome duplication occurred in the yeast Saccharomyces cerevisiae lineage and found compelling evidence for the existence of a contemporaneous interspecies hybridization. We propose that the genome doubling was a direct consequence of this hybridization and that it served to provide stability to the recently formed allopolyploid. This scenario provides a mechanism for the origin of this ancient duplication and the lineage that originated from it and brings a new perspective to the interpretation of the origin and consequences of whole-genome duplications.     

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution,Obtained Using Small-Angle X-Ray Scattering

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed.     

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius 〈R^MT〉 of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in 〈R^MT〉 seems to be isoform independent. Significantly, 〈R^MT〉 was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C_o^MT of MTs leading to changes in the curvature C^MT (=1/R^MT). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.     

Characterization of the Decaheme c-Type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-Ray Scattering and Neutron Reflectometry

The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (α-Fe₂O₃). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 × 90 × 65 ų. To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by ∼7 Å (the maximum dimension was 96 Å for oxidized OmcA, and 89 Å for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcA's role in mediating redox processes.     

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.     

Evidence for a Partially Structured State of the Amylin Monomer

Islet amyloid polypeptide (amylin) is the main component in amyloid deposits formed in type II diabetes. We used triplet quenching to probe the dynamics of contact formation between the N-terminal disulfide loop and a C-terminal tryptophan in monomeric amylins from human and rat. Quenching rates measured in the absence of denaturant are four times larger than those in 6 M guanidinium chloride, indicating a decrease in the average end-to-end distance (collapse) at low denaturant concentrations. We were surprised to find an even greater (sevenfold) increase in quenching rates on removal of denaturant for a hydrophilic control peptide containing the disulfide loop compared to the same peptide without the loop (twofold change). These results suggest that collapse is driven by backbone-backbone and backbone-side chain interactions involving the disulfide loop portion of the chain rather than by the formation of side-chain hydrophobic contacts. Molecular dynamics simulations of the control peptide show that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop. The quenching experiments also indicate that the monomer of the human, amyloidogenic form of amylin is more compact than the rat form, which does not form amyloid. We discuss these newly observed differences between human and rat amylin in solution and their possible relation to aggregation and to the physiological function of amylin binding to the calcitonin receptor.     

Formation of Iron Oxide Nanoparticles in the Internal Cavity of Ferritin-Like Dps Protein: Studies by Anomalous X-Ray Scattering

Biochemistry (Moscow) - DNA-binding protein from starved cells (Dps) takes a special place among dodecamer mini-ferritins. Its most important function is protection of bacterial genome from various...