The effect of lintnerisation on wheat and potato starch granules

Wheat and potato starches were hydrolysed with 2·2 n hydrochloric acid at 35°C for a period of time up to 15 days. The residues (lintnerised starches) were washed and freeze dried, and studied by differential scanning calorimetry (DSC), wide-angle X-ray scattering (WAXS), small-angle light scattering (SALS), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). These techniques showed that profound changes took place in the first day of hydrolysis (during which time the extent of hydrolysis was 7·7% for potato starch and 12·5% for wheat starch). In particular, the gelatinisation enthalpy (ΔH) decreased, the X-ray crystallinity increased and the SANS and SAXS peaks (indicative of a regular spacing between crystalline and amorphous regions) virtually disappeared. The reduction in ΔH is surprising and is discussed at length. It was also shown that freeze drying results in a considerable lowering of the gelatinisation temperature of potato starch (and also of ΔH) while that of wheat starch is only slightly affected.     

Dynamical dimer structure and liquid structure of fatty acids in their binary liquid mixture: dodecanoic and 3-phenylpropionic acids system

Dimer structure and liquid structure of fatty acids in the binary liquid mixture of dodecanoic (LA) and 3-phenylpropionic acids (PPA) were studied through the measurements of DSC, self-diffusion coefficient (D), density, viscosity, 13C NMR spin-lattice relaxation time, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS). The phase diagram of LA/PPA mixture exhibited a typical eutectic pattern, which means that LA and PPA are completely immiscible in solid phase. In the liquid phase of the LA/PPA mixture, D of LA always differed from that of PPA irrespective of their compositions. This exhibited that, in the liquid phase of the binary mixture of fatty acids giving a complete eutectic in the solid phase, the fatty acid dimers are composed of the same fatty acid species irrespective of their compositions. The liquid structure of the LA/PPA mixture was clarified through the SAXS and also the SANS measurements.     

Neutron and light scattering studies of light-harvesting photosynthetic antenna complexes

Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been employed in studying the structural information of various biological systems, particularly in systems without high-resolution structural information available. In this report, we briefly present some principles and biological applications of neutron scattering and DLS, compare the differences in information that can be obtained with small-angle X-ray scattering (SAXS), and then report recent studies of SANS and DLS, together with other biophysical approaches, for light-harvesting antenna complexes and reaction centers of purple and green phototrophic bacteria.     

Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study

Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 A and 19 A, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.     

Determination of asymmetric structure of ganglioside-DPPC mixed vesicle using SANS,SAXS, and DLS

Functions of mammalian cell membrane microdomains being rich in glycosphingolipids, so-called rafts, are now one of the current hot topics in cell biology from the intimate relation to cell adhesion and signaling. However, little is known about the role of glycosphingolipids in the formation and stability of the domains. By the use of the inverse contrast variation method in small-angle neutron scattering (SANS), combined with small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS), we have determined an asymmetric internal structure of the bilayer of the small unilamellar vesicle (SUV) of monosialoganglioside (G(M1))-dipalmitoylphosphatidylcholine (DPPC) mixture ([G(M1)]:[DPPC] = 0.1:1). A direct method using a shell-model fitting with a size distribution function describes consistently all experimental results of SANS, SAXS, and DLS. We have found that G(M1) molecules predominantly localize at SUV outer surface to form a highly hydrophilic layer which is dehydrated with the rise of temperature from 25 degrees C to 55 degrees C accompanied by the conformational change of the oligosaccharide chains. The average SUV size determined is approximately 200 A, which is comparable to the reported value 260 +/- 130 A of glycosphingolipids microdomains. The present results suggest that the preferential asymmetric distribution of gangliosides is essential to define the size and stability of the domains.     

Small-angle neutron scattering contrast variation studies of biological complexes: Challenges and triumphs

Small-angle neutron scattering (SANS) has been a beneficial tool for studying the structure of biological macromolecules in solution for several decades. Continued improvements in sample preparation techniques, including deuterium labeling, neutron instrumentation and complementary techniques such as small-angle x-ray scattering (SAXS), cryo-EM, NMR and x-ray crystallography, along with the availability of more powerful structure prediction algorithms and computational resources has made SANS more important than ever as a means to obtain unique information on the structure of biological complexes in solution. In particular, the contrast variation (CV) technique, which requires a large commitment in both sample preparation and measurement time, has become more practical with the advent of these improved resources. Here, challenges and recent triumphs as well as future prospects are discussed.     

Structural Studies of Overlapping Dinucleosomes in Solution

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In this study, the solution structure of the OLDN was investigated through the integration of small-angle x-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis and searched for the conformations that reproduced the SAXS and SANS scattering curves well. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.     

NMR and small-angle scattering-based structural analysis of protein complexes in solution

Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or residue-resolution structure determination and investigation of dynamic properties of multi-domain proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide.     

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.     

Selective deuteration of tryptophan and methionine residues in maltose binding protein: a model system for neutron scattering

We describe methods that have been developed within the ILL-EMBL Deuteration Laboratory for the production of maltose binding protein (MBP) that has been selectively labelled either with deuterated tryptophan or deuterated methionine (single labelling), or both (double labelling). MBP is used as an important model system for biophysical studies, and selective labelling can be helpful in the analysis of small-angle neutron scattering (SANS) data, neutron reflection (NR) data, and high-resolution neutron diffraction data. The selective labelling was carried out in E. coli high-cell density cultures using auxotrophic mutants in minimal medium containing the required deuterated precursors. Five types of sample were prepared and studied: (1) unmodified hydrogenated MBP (H-MBP), (2) perdeuterated MBP (D-MBP), (3) singly labelled MBP with the tryptophan residues deuterated (D-trp MBP), (4) singly labelled MBP with methionine residues deuterated (D-met MBP) and (5) doubly labelled MBP with both tryptophan and methionine residues deuterated (D-trp/met MBP). Labelled samples were characterised by size exclusion chromatography, gel electrophoresis, light scattering and mass spectroscopy. Preliminary small-angle neutron scattering (SANS) experiments have also been carried out and show measurable differences between the SANS data recorded for the various labelled analogues. More detailed SANS experiments using these labelled MBP analogues are planned; the degree to which such data could enhance structure determination by SANS is discussed.     

Small angle neutron and X-ray scattering in structural biology: recent examples from the literature

Small angle scattering can provide unique structural information on the shape, domain organisation, and interactions of biomacromolecules in solution. Small angle neutron scattering (SANS) combined with deuterium labelling makes it possible to define the positions of specific components within a complex while small angle X-ray scattering (SAXS) provides more precise data on the overall shape. Here I review four recent publications, three of which were presented at the Neutrons in Biology meeting at the STFC Rutherford Appleton Laboratory in July 2007, that utilise SANS, SAXS, and complementary techniques to define the solution structure of large multidomain proteins and macromolecular complexes. These four papers emphasise the critical importance of sample quality and characterisation as well as the important role played by complementary techniques in building structural models based on small angle scattering data. They show the ability of SANS and SAXS in determining solution structures provides an important complementary structural technique for large, flexible, and glycosylated proteins where high resolution structural techniques, such as crystallography and NMR, cannot be applied.     

Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex

Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.     

Kinetic asymmetry of subunit exchange of homooligomeric protein as revealed by deuteration-assisted small-angle neutron scattering

We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D₂O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.     

Derivation of the small-angle x-ray scattering functions for local conformations of polypeptide chains in solution

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.     

Small angle neutron scattering from lysozyme solutions in unsaturated and supersaturated states (SANS from lysozyme solutions)

Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.     

Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings

Solution structures of nucleosomes containing a human histone variant, H2A.Z.1, were measured by small-angle X-ray and neutron scatterings (SAXS and SANS). SAXS revealed that the outer shape, reflecting the DNA shape, of the H2A.Z.1 nucleosome is almost the same as that of the canonical H2A nucleosome. In contrast, SANS employing a contrast variation technique revealed that the histone octamer of the H2A.Z.1 nucleosome is smaller than that of the canonical nucleosome. The DNA within the H2A.Z.1 nucleosome was more susceptible to micrococcal nuclease than that within the canonical nucleosome. These results suggested that the DNA is loosely wrapped around the histone core in the H2A.Z.1 nucleosome.     

SAXS-data-based structural modeling of DNA–gadolinium complexes fixed in particles of cholesteric liquid-crystalline dispersions

Structure of cholesteric liquid-crystalline dispersions (CLCDs) formed by double-stranded DNA molecules and treated with gadolinium salts was studied by small-angle X-ray scattering (SAXS). The obtained SAXS data open the way for structural modeling of these complexes to obtain a reasonable explanation for the correlated decrease in amplitude of an abnormal negative band in the circular dichroism (CD) spectra and the characteristic Bragg peak in the experimental small-angle X-ray scattering curves observed on treatment of CLCD by gadolinium salts. Model simulations of different kinds of structural organizations of the DNA–gadolinium complex were performed using novel SAXS data analysis methods in combination with several new, complementary modeling techniques, enabling us to build low-resolution three-dimensional structural models of DNA–gadolinium complexes fixed in CLCD particles. The obtained models allow us to suggest that a change takes place in the helical twist of quasinematic layers formed by these molecules at high concentrations of gadolinium salt. This change in the twist can be used to explain the experimentally observed increase in amplitude of an abnormal band in the CD spectra of DNA CLCD.     

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.     

Quaternary structure built from subunits combining NMR and small-angle x-ray scattering data

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.