共查询到20条相似文献,搜索用时 31 毫秒
1.
Kozielski F Svergun D Zaccai G Wade RH Koch MH 《The Journal of biological chemistry》2001,276(2):1267-1275
The quaternary structures of several monomeric and dimeric kinesin constructs from Homo sapiens and Drosophila melanogaster were analyzed using small angle x-ray and neutron scattering. The experimental scattering curves of these proteins were compared with simulated scattering curves calculated from available crystallographic coordinates. These comparisons indicate that the overall conformations of the solution structures of D. melanogaster and H. sapiens kinesin heavy chain dimers are compatible with the crystal structure of dimeric kinesin from Rattus norvegicus. This suggests that the unusual asymmetric conformation of dimeric kinesin in the microtubule-independent ADP state is likely to be a general feature of the kinesin heavy chain subfamily. An intermediate length Drosophila construct (365 residues) is mostly monomeric at low protein concentration whereas at higher concentrations it is dimeric with a tendency to form higher oligomers. 相似文献
2.
Structural properties of rabbit skeletal myosin head (S1) and the influence of the DTNB light chain (LC2) on the size and shape of myosin heads in solution were investigated by small angle x-ray scattering. The LC2 deficient myosin head, S1 (-LC2), and the S1 containing LC2 light chain, S1 (+LC2) were studied in parallel. The respective values of the radius of gyration were found to be (40.2 +/- 0.5) A and (46.7 +/- 1) A, while the maximum dimension was (190 +/- 15) A for both species. The large difference between the two Rg values suggest that LC2 is located close to one extremity of the myosin head, in agreement with most electron microscopy observations. All models derived from the x-ray scattering pattern of the native myosin head share a common overall morphology, showing two main regions, an asymmetric globular portion which tapers smoothly into a thinner domain of roughly equivalent length making an angle of approximately 60 degrees, with a contour length of approximately 210 A. 相似文献
3.
The disulfide-reduced form of bovine ribonuclease A, with the Cys thiols irreversibly blocked, was characterized by small-angle x-ray scattering. To help resolve the conflicting results and interpretations from previous studies of this model unfolded protein, we measured scattering profiles using a range of solution conditions and compared them with the profiles predicted by a computational model for a random-coil polypeptide. Analysis of the simulated and experimental profiles reveals that scattering intensities at intermediate angles, corresponding to interatomic distances in the range of 5-20 Å, are particularly sensitive to changes in solvation and can be used to assess the internal scaling behavior of the polypeptide chain, expressed as a mass fractal dimension, Dm. This region of the scattering curve is also much less sensitive to experimental artifacts than is the very small angle regime (the Guinier region) that has been more typically used to characterize unfolded proteins. The experimental small-angle x-ray scattering profiles closely matched those predicted by the computational model assuming relatively small solvation energies. The scaling behavior of the polypeptide approaches that of a well-solvated polymer under conditions where it has a large net charge and at high urea concentrations. At lower urea concentrations and neutral pH, the behavior of the chain approaches that expected for θ-conditions, where the effects of slightly unfavorable interactions with solvent balance those of excluded volume, leading to scaling behavior comparable to that of an idealized random walk chain. Though detectable, the shift toward more compact conformations at lower urea concentrations does not correspond to a transition to a globule state and is associated with little or no reduction in conformational entropy. This type of collapse, therefore, is unlikely to greatly reduce the conformational search for the native state. 相似文献
4.
The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution. 相似文献
5.
Dario Braga Elena Ferracini Adele Ferrero Alberto Ripamonti David A. Brant Gregory S. Buliga Attilio Cesàro 《International journal of biological macromolecules》1985,7(3):161-166
An investigation of the small-angle X-ray scattering properties of aqueous solutions of an amylose derivative has been carried out. Experiments have been conducted in stable and fairly concentrated polymer solutions (up to 3.2%) by using a slightly substituted carboxymethylamylose having a degree of substitution of 0.08. Scattering intensities display a maximum in the low angle range which prevents extrapolation of the angular dependence to zero angle. Data obtained in the range of scattering vector 0.01<η<0.1Å?1 yield 8 Å as the radius of gyration of the chain cross-section and 140 dalton Å?1 as the mass per unit length. These results are analysed in terms of the current model of amylose solution conformation and compared with the theoretical calculations of the Debye scattering function of the isolated chain. 相似文献
6.
Protein interactions in undersaturated and supersaturated solutions: a study using light and x-ray scattering
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Protein interactions in undersaturated and supersaturated solutions were investigated using static and dynamic light scattering and small angle x-ray scattering. A morphodrom of lysozyme crystals determined at 35 degrees C and pH = 4.6 was used as a guideline in selecting the protein and precipitant concentrations. The osmotic second virial coefficient, B(22), was determined by static and dynamic light scattering. At low ionic strengths for which no crystals were formed, B(22) was positive indicating repulsive interactions between the protein molecules. Negative B(22) at higher ionic strengths corresponds to attractive interactions where crystallization becomes possible. At two extreme salt concentrations, small angle x-ray scattering data were collected and fitted with a statistical mechanical model based on Derjaguin-Landau-Verwey-Overbeek potential using Random Phase Approximation. This model accounted well for the small angle x-ray scattering data at undersaturated condition with constant potential parameters. At very high salt concentration corresponding to supersaturated solution this model seems to fail, possibly due to the presence of non-Derjaguin-Landau-Verwey-Overbeek hydration repulsion between the molecules. 相似文献
7.
If solution scattering curves can be accurately predicted from structural models, measurements can provide useful tests of predictions of secondary and tertiary structure. We have developed a computational technique for the prediction and interpretation of x-ray scattering profiles of biomolecules in solution. The method employs a Monte Carlo procedure for the generation of length distribution functions and provides predictions to moderate resolution (~5 Å). In addition to facilitating the assignment and interpretation of features in a solution scattering profile, the method also allows the elucidation of the role of protein motion in shaping the final scattering curve. The effect of protein motion on a scattering profile is investigated by generating scattering curves from several consecutive 0.147 ps atomic coordinate frames from a molecular dynamics simulation of the motion of bovine pancreatic trypsin inhibitor (BPTI) [McCammon, J. A. & Karplus, M. (1980) Annu. Rev. Phys. Chem. 31 , 29–45]. The theoretical approach is applied to chicken egg white lysozyme and BPTI, and the overall features in the resulting theoretical scattering profiles match well with the experimental solution scattering curves recorded on film. It is apparent from this study that the scattering profile prediction technique in conjunction with other experimental methods may have value in testing ideas of conformational change based on crystallographic studies; investigations of this type would include a comparison of predicted scattering curves generated from a variety of crystallographic models with an actual scattering profile of the biomolecule in solution. 相似文献
8.
H H Paradies 《The Journal of biological chemistry》1979,254(16):7495-7504
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. 相似文献
9.
Michael A. Jamros Leandro C. Oliveira Paul C. Whitford José N. Onuchic Joseph A. Adams Donald K. Blumenthal Patricia A. Jennings 《The Journal of biological chemistry》2010,285(46):36121-36128
C-terminal Src kinase (Csk) phosphorylates and down-regulates the Src family tyrosine kinases (SFKs). Crystallographic studies of Csk found an unusual arrangement of the SH2 and SH3 regulatory domains about the kinase core, forming a compact structure. However, recent structural studies of mutant Csk in the presence of an inhibitor indicate that the enzyme accesses an expanded structure. To investigate whether wt-Csk may also access open conformations we applied small angle x-ray scattering (SAXS). We find wt-Csk frequently occupies an extended conformation where the regulatory domains are removed from the kinase core. In addition, all-atom structure-based simulations indicate Csk occupies two free energy basins. These basins correspond to ensembles of distinct global conformations of Csk: a compact structure and an extended structure. The transitions between these structures are entropically driven and accessible via thermal fluctuations that break local interactions. We further characterized the ensemble by generating theoretical scattering curves for mixed populations of conformations from both basins and compared the predicted scattering curves to the experimental profile. This population-combination analysis is more consistent with the experimental data than any rigid model. It suggests that Csk adopts a broad ensemble of conformations in solution, populating extended conformations not observed in the crystal structure that may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the ubiquitous dynamic reversible assembly processes inherent in biology. 相似文献
10.
Apo- and holo-forms of horse liver alcohol dehydrogenase (LADH) in solution were studied by diffuse x-ray scattering. Experimental scattering curves for apo- and holo-forms coincide both with the curves calculated from the crystal structures of apo- and holo-enzymes, and with each other. Thus the “sliding” of catalytical domains in LADH upon substrate binding, which has been shown by x-ray analysis, cannot be detected by diffuse x-ray scattering. Sensitivity of the scattering curves to the domain displacements of sliding and “locking” types has been investigated. It has been shown that the scattering curves of LADH are rather sensitive to the domain “unlocking.” However, these curves change only slightly upon sliding of domains, including the sliding of domains observed in LADH by x-ray analysis. 相似文献
11.
Svergun DI Zaccai G Malfois M Wade RH Koch MH Kozielski F 《The Journal of biological chemistry》2001,276(27):24826-24832
The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5. 相似文献
12.
A method for determining transmembrane helix association and orientation in detergent micelles using small angle x-ray scattering.
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Solution small angle x-ray scattering can be used to study the association of transmembrane proteins solubilized in detergent micelles. We have used the alpha-helical transmembrane domain of the human erythrocyte glycophorin A (GpA) fused to the carboxyl terminus of monomeric staphylococcal nuclease (SN/GpA) as a model system for study. By matching the average electron density of the detergent micelles to that of the buffer solution, the micelle contribution to the small angle scattering vanishes, and the molecular weight and the radius of gyration of the proteins can be determined. SN/GpA has been found to dimerize in a zwitterionic detergent micelle, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), whose average electron density naturally matches the electron density of an aqueous buffer. The dimerization occurs through the transmembrane domains of GpA. With the aid of the nuclease domain scattering, the orientation of the helices within a dimer can be determined to be parallel by radius of gyration analysis. The association constant of a mutant (G83I) that weakens the GpA dimerization has been determined to be 24 microM in the DDMAB environment. The experimental methods established here could be used to apply solution small angle x-ray scattering to studying the association and interactions of other membrane proteins. 相似文献
13.
Tristan Turquois Cyrille Rochas Francois-R Taravel Jean Louis Doublier Monique-A-V. Axelos 《Biopolymers》1995,36(5):559-567
Small-angle x-ray scattering using a synchroton x-ray monochromatic radiation was carried out to investigate the structure of different polysaccharides in aqueous medium: carob galactomannan, κ-carrageenan in the sol and in the gel states, and κ-carrageenan-carob galactomannan mixed systems. Experiments performed on a 0.2% carob galactomannan solutions confirmed that this polysaccharide behaved as a neutral polymer in a good solvent. For K-carrageenan in the / state, either in the sodium form or in the cesium form, a maximum in the scattering curve was evidenced. Position and height of this maximum changed with K-carrageenan concentration in close agreement with what is expected for wormlike polyelectrolyte in semidilute solution. In the case of k-carrageenan in the gel state, in the cesium form, scattering curves also exhibited a maximum at an intermediate Q value. The position of this correlation peak did not change with concentration while its intensity increased. This effect was ascribed to a packing of rodlike structures by analogy with a suspension of colloidal elongated particles. This local structure could be viewed as bundles of parallel double helices. Addition of carobgalactomannan in κ-carrageenan gels induced dramatic structural changes. As the galactomannan concentration increased, the correlation peak tended to vanish. In contrast, no change in the cross-sectional radius of gyration was noticed. This phenomenon suggested a screening effect of the galactomannan, resulting in a loss of the correlation between the κ-carrageenan double helices. © 1995 John Wiley & Sons, Inc. 相似文献
14.
To explore the potential of utilizing Compton scattered x-ray photons for imaging applications, it is critical to accurately evaluate scattered x-ray transmission properties of targeted tissue materials. In this study, scattered x-ray transmission of breast tissue equivalent phantoms was evaluated. Firstly, two validations were carried out using a primary x-ray beam at 80 kVp with both experimental measurement (ion chamber with narrow-beam setup) and analytical calculation (Spektr toolkit). The tungsten-anode x-ray spectrum model was thus validated by measuring and calculating the transmission through increasing thickness of 1100 Aluminum filters. Similarly, the composition models of breast tissue equivalent phantoms (CIRS, 012A) were validated by measuring and calculating x-ray transmission for three different breast compositions (BR30/70, BR50/50, and BR70/30). Following validation, transmission properties of Compton scattered x-ray photons were measured with a GOS based linear array detector at the 90° angle from the primary beam. The same study was performed through three independent approaches: experimental measurement, analytical calculation, and Monte Carlo simulation (GEANT4). For all three methods, the scattered x-ray photon transmission as functions of phantom thickness were determined and fit into exponential functions. The transmission curves from all three methods matched reasonably well, with a maximum difference of 6.3% for the estimated effective attenuation coefficients of the BR50/50 phantom. The relative difference among the three methods of estimated attenuation is under 3.5%. As an initial step to develop a novel Compton scatter-based breast imaging system, the quantitative results from this study paved a fundamental base for future work. 相似文献
15.
K K Chohan M Jones J G Grossmann F E Frerman N S Scrutton M J Sutcliffe 《The Journal of biological chemistry》2001,276(36):34142-34147
Electron-transferring flavoproteins (ETFs) from human and Paracoccus denitrificans have been analyzed by small angle x-ray scattering, showing that neither molecule exists in a rigid conformation in solution. Both ETFs sample a range of conformations corresponding to a large rotation of domain II with respect to domains I and III. A model of the human ETF.medium chain acyl-CoA dehydrogenase complex, consistent with x-ray scattering data, indicates that optimal electron transfer requires domain II of ETF to rotate by approximately 30 to 50 degrees toward domain I relative to its position in the x-ray structure. Domain motion establishes a new "robust engineering principle" for electron transfer complexes, tolerating multiple configurations of the complex while retaining efficient electron transfer. 相似文献
16.
Hartmann H Lohkamp B Hellmann N Decker H 《The Journal of biological chemistry》2001,276(23):19954-19958
Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves. 相似文献
17.
The oligomeric states of bovine visual arrestin in solution were studied by small-angle x-ray scattering. The Guinier plot of arrestin at the concentration ranging from 0.4 mg/ml to 11.1 mg/ml was approximated with a straight line, and the apparent molecular weight was evaluated by the concentration-normalized intensity at zero angle (I(0)/conc). Using ovalbumin as a molecular weight standard, it was found that arrestin varied from monomer to tetramer depending on the concentration. The I(0)/conc decreased at high-salt concentration, but was independent of temperature. The simulation analysis of the concentration-dependent increase of I(0)/conc demonstrated that the tetramerization is highly cooperative, and arrestin at the physiological concentration is virtually in the equilibrium between monomer and tetramer. The concentration of arrestin monomer, which is considered to be an active form, remains at an almost constant level even if the total concentration of arrestin fluctuates within the physiological range. The scattering profile of arrestin tetramer in solution was in good agreement with that in the crystal, indicating that the quaternary structure in solution is essentially identical to that in crystal. Small-angle x-ray scattering was applied to a binding assay of phosphorylated rhodopsin and arrestin in the detergent system, and we directly observed their association as the increase of I(0)/conc. 相似文献
18.
Laboratory-made samples of the polysaccharide xylinan, also called acetan, were studied in aqueous solution at various ionic strengths I (0.01 mol/L ≤ I ≤ 0.30 mol/L). The conditions for clarification (ultracentrifugation/membrane filtration) were studied. The Zimm procedure was used to obtain the average molar mass, the second virial coefficient, and the radius of gyration. The interpretation of the angular dependence of scattered light by fitting with “Master Curves” led to double-stranded wormlike chains with persistence lengths between 90 and 120 nm. The ionic strength had a strong effect on the thermodynamic second virial coefficient, but the overall structure remained unchanged. The ambiguity of the light scattering data was discussed assuming alternatively a two-component system instead of the wormlike chain model for the experimental scattering curves. The two-component system can be ruled out on the basis of model calculations. © 1996 John Wiley & Sons, Inc. 相似文献
19.
Muroga Y 《Biopolymers》2001,59(5):320-329
The small-angle x-ray scattering (SAXS) functions are analytically derived for both the randomly coiled and helical local conformations of a polypeptide chain in solution. The resulting scattering functions for helices of various types are characterized by a maximum in the range of scattering-vector corresponding to Bragg spacings of 3-5 A, whereas the random-coil function has no maximum. This result is compatible with the extant SAXS data for partially neutralized poly(L-glutamic acid) and poly(L-lysine) in aqueous solutions. Comparison of the SAXS data with the calculated scattering functions shows that helical structures in both polypeptide chains are of the 3.6(13)-helix (alpha-helix) rather than 3.0(10)-type. 相似文献
20.
Michael D. L. Suits Benjamin Pluvinage Adrienne Law Yan Liu Angelina S. Palma Wengang Chai Ten Feizi Alisdair B. Boraston 《The Journal of biological chemistry》2014,289(39):27264-27277
For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl. 相似文献