Solution structure of phosphorylase kinase studied using small-angle X-ray and neutron scattering.

Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.     

Solution structure of human von Willebrand factor studied using small angle neutron scattering

von Willebrand factor (VWF) binding to platelets under high fluid shear is an important step regulating atherothrombosis. We applied light and small angle neutron scattering to study the solution structure of human VWF multimers and protomer. Results suggest that these proteins resemble prolate ellipsoids with radius of gyration (R(g)) of approximately 75 and approximately 30 nm for multimer and protomer, respectively. The ellipsoid dimensions/radii are 175 x 28 nm for multimers and 70 x 9.1 nm for protomers. Substructural repeat domains are evident within multimeric VWF that are indicative of elements of the protomer quarternary structure (16 nm) and individual functional domains (4.5 nm). Amino acids occupy only approximately 2% of the multimer and protomer volume, compared with 98% for serum albumin and 35% for fibrinogen. VWF treatment with guanidine.HCl, which increases VWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales <10 nm without altering protein R(g). Treatment of multimer but not protomer VWF with random homobifunctional linker BS(3) prior to reduction of intermonomer disulfide linkages and Western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable intermonomer non-covalent interactions within the multimer. Overall, multimeric VWF appears to be a loosely packed ellipsoidal protein with non-covalent interactions between different monomer units stabilizing its solution structure. Local, and not large scale, changes in multimer conformation are sufficient for ADAMTS-13-mediated proteolysis.     

Protein dynamics studied by neutron scattering

This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.     

Dynamic properties of an oriented lipid/DNA complex studied by neutron scattering

The formation of lipid-DNA (CL-DNA) complexes called lipoplexes, proposed as DNA vectors in gene therapy, is obtained by adding DNA to a solution containing liposomes composed of cationic and neutral lipids. The structural and dynamic properties of such lipoplexes are determined by a coupling between the electrostatic interactions and the elastic parameters of the lipid mixture. An attempt to achieve a better understanding of the structure-dynamics relationship is reported herein. In particular, an elastic neutron scattering investigation of DOTAP-DOPC (dioleoyl trimethylammonium propane-dioleoyl phosphatidylcoline) complexed with DNA is described. Proton dynamics in this oriented CL-DNA lipoplex is found to be strongly dependent upon DNA concentration. Our results show that a substantial modification of the membrane dynamics is accompanied by the balancing of the total net charge inside the complex, together with the consequent displacement of interlayer water molecules.     

Hydration structure in natural DNA observed by thermal neutron scattering

Films of highly oriented Na- and LiDNA showing the typical X-ray diffraction patterns for the A-,B-, and C-conformation have been investigated by elastic and quasielastic neutron scattering. Information concerning the question of the DNA-water interaction has been obtained by varying the parameters H₂O/D₂O contrast, humidity, and temperature. Main observations are: A coexistence of one- and three-dimensionally correlated DNA which shifts towards the one-dimensionally correlated C-conformation for high humidity; a coexistence of A-, B-, and C-conformation for NaDNA with a similar humidity dependence; a factor of two increase between the average degree of localization of water hydrogens compared with DNA hydrogens at 75% r.h. for NaDNA; a strong water contribution to layer peaks which are close to the susceptibility maximum of water; a strong temperature dependence of the axial repeat distance for C-DNA; broad quasielastic spectra around the inverse of this distance. The observations are interpreted in terms of a competition between finite three-dimensional correlation and an optimized spatial resonance of nearly one-dimensionally correlated DNA with the correlation of bulk water. The observations are compatible with the concept of water spine formation (Dickerson 1983). The interpretation emphasizes the dynamic character of this mechanism in the region of nearly one-dimensionally correlated DNA.     

Mechanism of force generation studied by neutron scattering

Neutron scattering has been used to compare the structure of myosin S1 that is free in solution to that when it is bound to F-actin. To achieve this, deuterated actin was obtained from D. discoideum that had been fed deuterated E. coli. This deuterated actin was rendered “invisible” to neutrons when dissolved in 94% D₂O. The neutron scattering patterns obtained from S1 bound to deuterated actin were identical to those of free S1 except for oscillations due to S1's bound to the same actin filament. At low S1 to actin stoichiometrics, these oscillations diminish and the patterns become indistinguishable. The apparent radius of gyration of S1 bound to actin is identical to that of free S1 when the stoichiometry is low. Thus, no changes in the structure of S1 were observed to a resolution of 2.5 nm. Computer modelling studies were used to evaluate the compatibility of models for the mechanism of force generation with the neutron data. These studies show that for powerstrokes greater than 5.0 nm, the data are consistent with more than 80% of the crossbridge maintaining a rigid conformation during force generation.     

Kappa-casein micelles: structure, interaction and gelling studied by small-angle neutron scattering

Small-angle neutron scattering (SANS) measurements on dilute and concentrated dispersions of kappa-casein micelles in a buffer at pH = 6.7 were made using the D11 diffractometer in Grenoble. Results indicate that the micelles have a dense core with a fluffy outer layer. This outer layer appears to give rise to a steeply repulsive interaction on contact. In fact, the hard-sphere model best fits the measured scattering intensities. Adding chymosin to the dispersion initiated a fractal flocculation of the micelles and consecutively a coalescence of the micelles. This unexpected second process resembled that of spinodal demixing. The dispersion phase thus separates into a water and a protein phase on a time scale of hours. The observed phenomona contribute to the understanding of the cheese-making process.     

Structure of casein micelles studied by small-angle neutron scattering

The structure of casein micelles has been studied by small-angle neutron scattering and static light scattering. Alterations in structure upon variation of pH and scattering contrast, as well as after addition of chymosin, were investigated. The experimental data were analyzed by a model in which the casein micelle consists of spherical submicelles. This model gave good agreement with the data and gave an average micellar radius of about 100–120 nm and a submicellar radius of about 7 nm both with a polydispersity of about 40–50%. The contrast variation indicated that the scattering length density of the submicelles was largest at the center of the submicelles. The submicelles were found to be closely packed, the volume fraction varying slightly with pH. Upon addition of chymosin the submicellar structure remained unchanged within the experimental accuracy. Correspondence to: S. Hansen     

beta-lactoglobulin under high pressure studied by small-angle neutron scattering

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.     

Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram

Small-angle neutron scattering has been used to study structural features of lamellar bilayer membranes of dimyristoylphosphatidylcholine (DMPC) and DMPC mixed with various amount of cholesterol. The studies were recorded at a fixed hydration level of 17% 2H2O, i.e. just below saturation. Bragg reflections gives information on the ripple structure and on the bilayer periodicity. The crystalline Lc phase, which was stabilized after long time storage at low temperature, exhibits major small angle scattering when cholesterol is mixed into the membrane. The intermediate P beta' gel-phase, which is characteristic by the rippled structure, is dramatically stabilized by the introduction of cholesterol. The ripple structure depends significantly both on the cholesterol content and on the temperature. At high temperatures, T greater than 15 degrees C, the inverse ripple periodicity varies basically linearly with cholesterol content, and approach zero (i.e. periodicity goes to infinite) at 20 mol% cholesterol, approximately. At lower temperatures the correlation is more complex. The data indicate additional phase boundaries below 2 mol% and at approx. 8 mol%. Secondary rippled structures are observed in the low temperature L beta'-phase for cholesterol content below approx. 8 mol%. The data gives detailed insight into the phosphatidylcholine cholesterol phase diagram, which is discussed on the basis of a simple model in which the cholesterol complexes are fixed to the defect stripes of the rippled structure.     

Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations.

Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.     

Salt-dependent compaction of di- and trinucleosomes studied by small-angle neutron scattering

Using small-angle neutron scattering (SANS), we have measured the salt-dependent static structure factor of di- and trinucleosomes from chicken erythrocytes and from COS-7 cells. We also determined the sedimentation coefficients of these dinucleosomes and dinucleosomes reconstituted on a 416-bp DNA containing two nucleosome positioning sequences of the 5S rDNA of Lytechinus variegatus at low and high salt concentrations. The internucleosomal distance d was calculated by simulation as well as Fourier back-transformation of the SANS curves and by hydrodynamic simulation of sedimentation coefficients. Nucleosome dimers from chicken erythrocyte chromatin show a decrease in d from approximately 220 A at 5 mM NaCl to 150 A at 100 mM NaCl. For dinucleosomes from COS-7 chromatin, d decreases from 180 A at 5 mM to 140 A at 100 mM NaCl concentration. Our measurements on trinucleosomes are compatible with a compaction through two different mechanisms, depending on the salt concentration. Between 0 and 20 mM NaCl, the internucleosomal distance between adjacent nucleosomes remains constant, whereas the angle of the DNA strands entering and leaving the central nucleosome decreases. Above 20 mM NaCl, the adjacent nucleosomes approach each other, similar to the compaction of dinucleosomes. The internucleosomal distance of 140-150 A at 100 mM NaCl is in agreement with distances measured by scanning force microscopy and electron microscopy on long chromatin filaments.     

Formation of actin dimers as studied by small angle neutron scattering

Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization.     

The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering

The aggregation behavior of zinc-free insulin has been studied by small-angle neutron scattering as a function of pH and ionic strength of the solution. The pair distance distribution functions for the 12 samples have been obtained by indirect Fourier transformation. The results show that the diameter of the aggregates is 40 Å at pH 11 and 10 mM NaCl, independent of the protein concentration. The largest diameter of about 120 Å is found for pH 8, 100 mM NaCl, and a protein concentration of 10 mg/ml. Estimates of the pair distance distribution functions, free of inter-particle correlation effects, were obtained by an indirect Fourier transformation, omitting the data at small scattering vectors, which are influenced by these effects. By this procedure the weight-averaged molecular mass and the average radius of gyration were determined. These parameters vary from 1.3 times the monomer mass and 14 Å, to 6.8 times the monomer mass and 31 Å, respectively. The mass distribution between the oligomers was determined by a model based on the crystal structure of zinc-free insulin. The results from this model and the Fourier transformations have been compared to an equilibrium model recently introduced by Kadima et al. (1993). The neutron scattering results agree well with the predictions of this model except that broader mass distributions are suggested by neutron scattering. Correspondence to: J. Skov Pedersen     

Water structure around a left-handed Z-DNA fragment analyzed by cryo neutron crystallography

Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]₂. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.     

Rigidity of protein structure revealed by incoherent neutron scattering

The rigidity and flexibility of a protein is reflected in its structural dynamics. Studies on protein dynamics often focus on flexibility and softness; this review focuses on protein structural rigidity. The extent of rigidity can be assessed experimentally with incoherent neutron scattering; a method that is complementary to molecular dynamics simulation. This experimental technique can provide information about protein dynamics in timescales of pico- to nanoseconds and at spatial scales of nanometers; these dynamics can help quantify the rigidity of a protein by indices such as force constant, Boson peak, dynamical transition, and dynamical heterogeneity. These indicators also reflect the rigidity of a protein's secondary and tertiary structures. In addition, the indices reveal how rigidity is influenced by different environmental parameters, such as hydration, temperature, pressure, and protein-protein interactions. Hydration affects both rigidity and softness more than other environmental factors. Interestingly, hydration affects harmonic and anharmonic motions in opposite ways. This difference is probably due to the protein's dynamic coupling with water molecules via hydrogen bonding.