Conformation of poly(sodium ethacrylate) in solution studied by small-angle X-ray scattering

The pH-induced conformational transition of poly(sodium ethacrylate) PNaEA in aqueous solution, which occurs between a compact form at low charge-density and an extended coil at high charge-density, was studied by small-angle X-ray scattering and the structure at an each conformational state was analyzed and compared with the corresponding one of poly(sodium methacrylate) PNaMA. The conformational transition for PNaEA induced a remarkable change in the scattering data plotted in the form of the Kratky plot. By comparing the scattering data with theoretical scattering functions, it was clarified that the structures of the compact form and the extended coil are well mimicked by a swollen gel having a network structure and by a wormlike chain, respectively. Although such a structure of the extended coil of PNaEA is similar to the corresponding one of PNaMA, the structure of the compact form of PNaEA is different from the corresponding one of PNaMA, which is still represented by a wormlike chain in a Theta medium.     

Conformation study of poly(γ-methyl-L-glutamate) in helicogenic solvents by small-angle X-ray scattering

Small-angle x-ray scattering of poly(γ-methyl-L -glutamate), [Glu(OMe)]_n, in m-cresol and in pyridine was measured to determine the mass per unit length, M_q, and the radius of gyration of the cross section, 〈S〉^1/2. It was confirmed from the values of M_q that [Glu(OMe)]_n exists in an α-helical conformation in these solvents. It was elucidated from the calculations on 〈S〉^1/2 that the side chains come in moderately close contact with the main chain in these solvents. It was indicated from the analysis of the outer portion of the scattering curves that the side-chain conformation varied depending on the solvent.     

Conformation study on heparin by intermediate-angle X-ray scattering

The conformation of heparin in water was investigated by intermediate-angle x-ray scattering (IAXS). The theoretical scattering function for the coil conformation was calculated by the Monte Carlo method using the approximation of separable conformation energies and the conformation energies computed for two disaccharide pairs in heparin. From x-ray scattering in a relatively small-angle region, the conformation of heparin is not the ordered 2₁ helix conformation but the coil conformation obtained by the Monte Carlo calculation. It is expected, from x-ray scattering in a relatively wide-angel region, that the sulfate groups of heparin maintain about 7 Å between them.     

A study of an infected bone tissue (osteomyelitis) by small-angle X-ray scattering

The small-angle X-ray scattering method has been applied to evaluate various macromolecular parameters such as the specific inner surface, the transversal lengths, the length of crystallinity, the range of amorphous zone and the percentage of porosity in pure human bone and osteomyelitis, an infection of bone tissue. The hydroxyapatite crystals of bone being uniformly dispersed throughout the hydrated collagenous matrix creating a large mineral matrix interface, we found the bone samples to behave as a densely packed two phase system. The theories of Kratky and Porod have been utilized to evaluate the macromolecular parameters. These findings may shed light on tertiary structural deformation of human bone when it is infected.     

Conformation of poly(methacrylic acid) in acidic aqueous solution studied by small angle X-ray scattering

The nature of the contracted form of poly(methacrylic acid) PMA chain in salt-free acidic aqueous solution was studied by analyzing scattering curves registered by small-angle X-ray scattering, comparing it with those of PMA in methanol at 26 degrees C and of partially neutralized PMA in aqueous solution containing added salt (the concentration of added salt, Cs=0.1 M NaF). It is shown that the distribution of segments in the contracted form as well as that of PMA in methanol is that of a random-coil in a theta medium and that this distribution of segments is stable over a fair range of degrees of ionization alpha for Cs below 0.1 M. Moreover, the persistence length of PMA at Cs=0.1 M (4+/-0.5 A) is substantially constant throughout the entire range of alpha, indicating that the contracted form of PMA changes to an expanded random-coil in a higher pH region without a significant change in the chain flexibility.     

Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.     

Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.     

A small-angle X-ray solution scattering study of bovine alpha-crystallin.

The native high molecular mass form of alpha-crystallin, the most important soluble protein in the eye lens, and its low molecular mass form obtained at 37 degrees C in dilute solutions were investigated by synchrotron radiation small-angle X-ray scattering. The alpha-crystallin solutions are polydisperse and good fits to the experimental data can be obtained using distributions of spheres with radii varying between about 5 and 10 nm. In spite of the polydispersity, two different ab initio methods were used to retrieve low resolution shapes from the scattering data. These shapes correspond to the z-average structure of the oligomers. In the absence of any symmetry constraints, the scattering curves of the two forms of alpha-crystallin yield bean-like shapes. The shape corresponding to the low molecular mass form has about 20% less mass at the periphery. Imposing tetrahedral symmetry on the average structures worsens the fit to the experimental data. We emphasized the apparent contradiction between hydrodynamic and molecular properties of alpha-crystallin. An explanation was put forward based on the presence of solvent-exposed flexible C-terminal extensions. We present two bead models ('hollow globule with tentacles' and 'bean with tentacles') based on NMR and cryo-electron microscopy studies and discuss how well they correspond with our data from X-ray scattering, light scattering and analytical ultracentrifugation.     

Amylose conformation in aqueous solution: a small-angle X-ray scattering study

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.     

Micellar structure of beta-casein observed by small-angle X-ray scattering

The small-angle X-ray scattering was observed from beta-casein micelles in 0.2 M phosphate buffer (pH 6.7) with varying temperatures. An oblate ellipsoid of a rigid core with a thin soft layer was proposed as a probable model of the beta-casein micellar structure, according to the results of the model optimization with simple triaxial bodies. Here the axial ratio was found to decrease and the micelle to become spherical when the polymerization proceeds with temperature. The consistency of the present model was examined with the results of hydrodynamic measurements published previously.     

Uncoupler-induced changes in mitochondrial structure detected by small-angle X-ray scattering

Small-angle X-ray scattering data suggest that major but reversible rearrangements of mitochondrial inner membrane structure are induced by uncouplers. Low levels of 2,4-dinitrophenol (10 μM) cause a perceptible wide-angle shift of the 20 mrad X-ray scattering maximum characteristic of intact liver mitochondria. Higher dinitrophenol concentrations (> 25 μM) reduce this scattering maximum to one-third its initial intensity. In terms of mitochondrial function, the former scattering change appears to correlate with the uncoupling of oxidative phosphorylation while the latter occurs in the course of dinitrophenol stimulation of mitochondrial ATPase activity.     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

Interactions of hydrophobin proteins in solution studied by small-angle X-ray scattering

Hydrophobins are a group of very surface-active, fungal proteins known to self-assemble on various hydrophobic/hydrophilic interfaces. The self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins are also soluble in water. Here, the association of hydrophobins HFBI and HFBII from Trichoderma reesei in aqueous solution was studied using small-angle x-ray scattering. Both HFBI and HFBII exist mainly as tetramers in solution in the concentration range 0.5-10 mg/ml. The assemblies of HFBII dissociate more easily than those of HFBI, which can tolerate changes of pH from 3 to 9 and temperatures in the range 5°C-60°C. The self-association of HFBI and HFBII is mainly driven by the hydrophobic effect, and addition of salts along the Hofmeister series promotes the formation of larger assemblies, whereas ethanol breaks the tetramers into monomers. The possibility that the oligomers in solution form the building blocks of the self-assembled film at the air/water interface is discussed.     

Nonuniformity in natural rubber as revealed by small-angle neutron scattering, small-angle X-ray scattering, and atomic force microscopy

The microscopic structures of natural rubber (NR) and deproteinized NR (DPNR) were investigated by means of small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM). They were compared to those of isoprene rubber (IR), which is a synthetic analogue of NR in terms of chemical structure without any non-rubber components like proteins. Comparisons of the structure and mechanical properties of NR, DPNR, and IR lead to the following conclusions. (i) The well-known facts, for example, the outstanding green strength of NR and strain-induced crystallization, are due not much to the presence of proteins but to other components such as the presence of phospholipids and/or the higher stereoregularity of NR. It also became clear the naturally residing proteins accelerate the upturn of stress at low strain. The protein phases work as cross-linking sites and reinforcing fillers in the rubbery matrix. (ii) The microscopic structures of NR were successfully reproduced by SANS intensity functions consisting of squared-Lorentz and Lorentz functions, indicating the presence of inhomogeneities in bulk and thermal concentration fluctuations in swollen state, respectively. On the other hand, IR rubbers were homogeneous in bulk. (iii) The inhomogeneities in NR are assigned to protein aggregates of the order of 200 A or larger. Although these aggregates are larger in size as well as in volume fraction than those of cross-link inhomogeneities introduced by cross-linking, they are removed by deproteinization. (iv) Swelling of both NR and IR networks introduces gel-like concentration fluctuations whose mesh size is of the order of 20 A.     

The complete VS ribozyme in solution studied by small-angle X-ray scattering

We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of trans-esterification in which adenine and guanine nucleobases contributed by the interacting loops combine in general acid-base catalysis.     

pH-dependent unfolding of aspergillopepsin II studied by small-angle X-ray scattering

Aspergillopepsin II (EC 3.4.23.6) secreted from the fungus Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase. It consists of two polypeptide chains (i.e., a heavy chain and a light chain), which are bound noncovalently to each other. The pH titration analysis using small-angle X-ray scattering (SAXS) as well as circular dichroism (CD) and gel filtration indicated that the enzyme was unfolded around a neutral pH with concomitant dissociation of the two chains. Detailed analyses showed that the midpoint pH values for the unfolding are not coincident with one another (pH 6.1 in circular dichroism and gel filtration, pH 6.4 in zero-angle intensity of SAXS, pH 6.8 in radius of gyration). The difference between these values suggested the existence of an intermediate state during the unfolding. Further analyses of the SAXS data showed that the heavy chain just after the dissociation still kept molecular compactness and that it gradually increased its dimensions as the pH was further raised. Noncoincidence of the two phenomena (i.e., chain dissociation and swelling) led to elucidation of a novel intermediate state during unfolding, which was confirmed by the subsequent singular value decomposition (SVD) analysis.     

Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering

Small-angle scattering of X-rays (SAXS) is an established method to study the overall structure and structural transitions of biological macromolecules in solution. For folded proteins, the technique provides three-dimensional low resolution structures ab initio or it can be used to drive rigid-body modeling. SAXS is also a powerful tool for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs), and is highly complementary to the high resolution methods of X-ray crystallography and NMR. Here we present the basic principles of SAXS and review the main approaches to the characterization of IDPs and flexible multidomain proteins using SAXS. Together with the standard approaches based on the analysis of overall parameters, a recently developed Ensemble Optimization Method (EOM) is now available. The latter method allows for the co-existence of multiple protein conformations in solution compatible with the scattering data. Analysis of the selected ensembles provides quantitative information about flexibility and also offers insights into structural features. Examples of the use of SAXS and combined approaches with NMR, X-ray crystallography, and computational methods to characterize completely or partially disordered proteins are presented.