Structure of cross-linked rabbit muscle phosphofructokinase in solution.

Cross-linked rabbit muscle phosphofructokinase in the active tetrameric and octameric state was studied in solution by hydrodynamic methods and small angle x-ray scattering techniques. The translational diffusion coefficients were determined by means of inelastic light scattering and were found to be 3.60 (+/- 0.02) x 10(-7) cm2 . s-1 for the tetramer and 2.54 (+/- 0.15) x 10(-7) cm2 . s-1 for the octamer. From small angle x-ray scattering measurements the radius of gyration, the specific inner surface area, and the volume were determined for both enzyme forms, revealing that the octameric cross-linked form is approximately spherical, with a diameter of 120.0 A, whereas the tetrameric form is asymmetric having an axial ratio of 2. By comparison of the scattering curves with triaxial geometric bodies which are equivalent in scattering, the tetrameric enzyme is described as a rectangular prism, with overall dimensions of A = 131.0 A, B = 131.0 A, and C = 65.0 A, and the octameric form as that of a cube with A = B = C = 120.0 A. The shape of the protomer, having a radius of gyration of 24.8 A, in the tetramer and octamer is similar to that for the native tetramer at pH 10 in the presence of 5 mM fructose 6-phosphate or 15 mM fructose 1,6-bis-phosphate. From the different shapes of the scattering curves of the native phosphofructokinase at pH 7.5 in the presence of 15 mM ATP and of the cross-linked tetramer or octamer, it can be inferred that the shapes of the protomers are different: in the presence of ATP the protomers are elongated, having an axial ratio of 1.8 to 2.0; the cross-linked state reveals a spherical protomer of radius 33.0 A, similar to that of the native enzyme at pH 7.5 in the presence of fructose 6-phosphate or fructose 1,6-bisphosphate.     

Study of the substrate-induced changes in the state of Eco dam methylase using a method of small-angle x-ray scattering

Interaction of Ecodam methylase (E.C. 2.1.1) with synthetic oligonucleotide substrates of various primary structure was studied by the small angle X-ray scattering method. Complex formation between the enzyme and substrates occurs after addition of double-stranded oligonucleotides to the methylase. In the presence of 1 M NaC1 (when the enzyme is inactive) addition of the synthetic substrates does not result in complex formation. Comparison of the experimental scattering parameters with the calculated ones has been made. The best coincidence of these data is obtained for the model which proposed Ecodam methylase dimer formation in the course of its interaction with the substrates.     

Structural flexibility of the N-terminal beta-barrel domain of 15-lipoxygenase-1 probed by small angle X-ray scattering. Functional consequences for activity regulation and membrane binding

Mammalian lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases (atherosclerosis, osteoporosis) with major health political importance. The crystal structures of two plant lipoxygenase isoforms have been solved and X-ray coordinates for an inhibitor complex of the rabbit 15-lipoxygenase-1 are also accessible. Here, we investigated the solution structure of the ligand-free rabbit 15-lipoxygenase-1 by small angle X-ray scattering. From the scattering profiles we modeled the solution structure of the enzyme using two independent ab initio approaches. Preliminary experiments indicated that at low protein concentrations (<1mg/ml) and at 10 degrees C the enzyme is present as hydrated monomer. Superposition of the high resolution crystal structure and our low resolution model of the solution structure revealed two major differences. (i) Although the two models are almost perfectly superimposed in the region of the catalytic domain the solution structure is stretched out in the region of the N-terminal beta-barrel domain and exhibits a bigger molecular volume. (ii) There is a central bending of the enzyme molecule in the solution structure, which does not show up in the crystal structure. Both structural peculiarities may be explained by a high degree of motional freedom of the N-terminal beta-barrel domain in aqueous solutions. This interdomain movement may be of functional importance for regulation of the catalytic activity and membrane binding.     

X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin.

A protease inhibitor from hagfish blood plasma, homologous to human alpha 2-macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods.     

Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function

Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.     

Small angle X-ray scattering study on bovine porphobilinogen synthase (5-aminolaevulinate dehydratase)

The quaternary structure of the native (zinc) porphobilinogen synthase (5-amino-laevulinate dehydratase) from bovine liver and its lead-substituted derivative is studied in solution by small angle X-ray scattering. In spite of the profound inhibitory effect of lead ions in the enzyme they do not produce a change in the quaternary structure detectable by small angle X-ray scattering. The most important molecular parameters of the native enzyme were found to be: radius of gyration Rg = 4.04 +/- 0.04 nm and maximum dimension Dmax = 12.0 +/- 0.5 nm. The corresponding values for the lead derivative are: Rg = 4.26 +/- 0.1 nm and Dmax = 12.5 +/- 0.5 nm. The quaternary structure of the enzyme in solution is described by a model, which fits the experimental scattering and distance distribution function.     

Fully reduced ribonuclease A does not expand at high denaturant concentration or temperature

The dimensions of a denatured protein, fully reduced ribonuclease A (r-RNase A), have been measured using synchrotron-based small angle X-ray scattering. The radius of gyration, 34-35 A, is unchanged from 0-6 M guanidinium chloride and from 20-90 degrees C at pH 2.5, and agrees with the known scaling behavior for a multitude of chemically denatured states. The polypeptide is behaving as a statistical coil in the non-interacting, high-temperature limit.     

Structural properties of a monobrominated analog of 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine

Hydrated multibilayers of 1-palmitoyl-2-monobromopalmitoyl-sn-glycero-3-phosphorylcholine (BrDPPC), where the 2-chain is brominated at either the C-9 or C-10 position, have been studied by low and wide angle X-ray diffraction methods. Oriented and unoriented samples were investigated. The long spacing was observed over the temperature interval -15 degrees C to 80 degrees C. A monotonic increase from approx. 50 A to approx. 62 A (28 wt. % H2O) occurred with decreasing temperature. The BrDPPC showed no evidence of a sharp gel-to-liquid crystal phase transition. Wide angle scattering showed a diffuse peak corresponding to (4.5 A)-1. Differential scanning calorimetry measurements for hydrated liposomes (50 wt. % H2O) also showed no evidence for a phase transition (-40 less than or equal to T less than or equal to 60 degrees C). These results suggest a low temperature amorphous (glass) state for the acyl side chains of BrDPPC. Monolayer film properties of monobrominated stearic acid also reflect a chain disordering effect occurring upon midchain substitution.     

Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.     

Large size fibrillar bundles of the Alzheimer amyloid β-protein

Self-assembly of amyloid β-protein (Aβ) and its deposition into senile plaques are distinctive features of Alzheimer’s disease. Aβ forms typical linear aggregates known as amyloid fibrils, with a diameter of a few tens of nanometers and a length spanning from hundreds of nanometers to micrometers. Fibrils eventually assemble into large size clusters and precipitate in vivo in the brain deposits. Here, we study the late stage of aggregation of Aβ(1–40) in vitro at pH 3.1. We characterize the structure of fibrillar aggregates by a combined use of different experimental techniques. Small angle light scattering, heterodyne near field scattering, large angle light scattering, ultra small angle X-ray scattering and small angle X-ray scattering measurements have been performed to highlight the structural features of amyloid bundles over several lengthscales, from nanometers to tens of micrometers. Phase contrast optical microscopy has been used to complement scattering measurements and directly visualize some morphological details. We show that elongated fibrils of Aβ with a diameter of a few nanometers are packed into large size compact bundles having a typical size of tens of micrometers. The linear morphology of fibrils is reflected in the elongated shape of bundles. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Small angle x-ray scattering studies on adenovirus type 2 hexon

Adenovirus type 2 hexons have been studied in solution by small angle X-ray scattering, and the following molecular parameters determined: radius of gyration (Rg) = 4.9 nm, molecular weight (M) = 310.000, invariant volume (Vinv) = 630 mn3, maximal distance (Dmax) = 14.5--15.5 nm. A diffraction pattern was obtained up to an angular increment of h = 2.5 nm-1. Various models for the hexon have been explored by calculating the diffraction pattern from the Debye formula for 1200 spheres arranged to define the scattering volume of each model. Models were first built according to electron micrographic results. Later, preliminary results of a crystallographic study were used for model building. The experimental pattern and the pattern resulting from the model determined by crystallographic methods were compared and showed good agreement.     

Structural characterization of the nonameric assembly of an Archaeal alpha-L-fucosidase by synchrotron small angle X-ray scattering

alpha-l-Fucosidase is a lysosomal enzyme responsible for hydrolyzing the alpha-1,6-linked fucose joined to the reducing-end N-acetylglucosamine of carbohydrate moieties in glycoproteins. The first alpha-l-fucosidase from Archaea was recently identified in the genome of the hyperthermophile Sulfolobus solfataricus; the enzyme is encoded by two open reading frames separated by a -1 frameshift. A preliminary biochemical and biophysical characterization of this extremophile enzyme has been carried out both in solution, through small angle X-ray scattering experiments, and in the crystalline state, showing an unusual oligomeric assembly resulting from the association of nine subunits, endowed with 3-fold molecular symmetry.     

Small-angle X-ray scattering study of the (Fab′)2 fragment of the human immunoglobulin Kol

The conformation of the (Fab′)₂ fragment of the human immunoglubulin Kol has been investigated in solution by small angle X-ray scattering. The following molecular parameters were determined: radius of gyration 4.10 ± 0.05 nm; maximum distance 14.0 ± 0.5 nm and hydrated volume 150 ± 8 nm³. A model of the fragment is presented, which fits these experimental data and shows good agreement with the distance distribution function in real space and the scattering curve in reciprocal space. We have to assume that the (Fab′)₂ fragment has many different conformations in solution. The method of small-angle X-ray scattering only allows the determination of an average conformation which is very similar within the resolution of the method to the static structure determined in the crystal.     

Extended X-ray absorption fine structure study of the coupled binuclear copper active site of tyrosinase from Neurospora crassa

Cu K-edge X-ray absorption spectra have been recorded for the enzyme tyrosinase from Neurospora crassa, in its oxy, resting (met-aquo), and inhibitor-bound (met-mimosine) forms. The K-edges proper resemble those of oxy- and met-hemocyanin, and confirm the presence of CuII. The forbidden 1s----3d transition is noticeably stronger for the 1-mimosine-bound enzyme, implying some distortion of the tetragonal Cu coordination group on inhibitor binding. The extended fine structure (EXAFS) beyond the K-edge has been analyzed. The first shell scattering is consistent with the presence of two N- and two O-ligand atoms, at 2.0 and 1.9 A, for all three forms of the enzyme; there is no evidence for heavy atom (S) scattering in the first shell. As in analogous hemocyanin derivatives, the outer shell scattering contains contributions from distant atoms of imidazole ligands, as well as from an addition scattering atom, at 3.4-3.6 A. For oxy-tyrosinase the additional scatterer is unambiguously a heavy atom (Cu), although a larger Debye-Waller factor suggests a somewhat less rigid binuclear site than in oxy-hemocyanin.     

Crystallization and preliminary characterization of three crystal forms of human recombinant transforming growth factor-alpha.

Three crystal forms of human recombinant TGF-alpha have been grown from solutions containing 2-methyl-2,4-pentanediol. One of the forms belongs to the orthorhombic space group C222(1) and the other two belong to the monoclinic space group C2. Two of the crystal forms diffract to approximately 2.3 A Bragg spacings. X-ray diffraction data has been collected for all three forms. These data appear to be suitable for crystal structure determination, using either heavy atom isomorphous replacement methods or molecular replacement, for phase determination.     

Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.     

Solution structure of RNase P RNA

The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.     

X-ray small angle scattering study of chromatin as a function of fiber length

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.     

Crystal structure of an anti-Lewis alpha Fab determined by molecular replacement methods

The anti-Lewis alpha mouse immunoglobulin CF4C4 (IgGl, k) Fab has been crystallized from 58% saturated ammonium sulfate in space group Pl; unit cell dimensions a = 43.4 A b = 41.7 A, c = 62.0 A, a = 72.7 degrees, beta = 96.6 degrees, gamma = 100.1 degrees. X-ray diffraction data have been measured beyond 3.0 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using as search models the constant and variable domains of the mouse immunoglobulin McPC603 (IgA, kappa) Fab. The crystallographic residual for the data 5.0 to 4.0 A, is 0.47. The approximate 2-fold axis relating the VL and the VH domains forms an angle of 164 degrees with the 2-fold axis relating the constant domains. The crystal packing is reasonable.