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PubMed1992

Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering.

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.

Complement C9↗
PubMed1980

The subunit positions within RNA polymerase holoenzyme determined by triangulation of centre-to-centre distances.

The complete 'centre-of-subunit structure' of the multisubunit enzyme DNA-dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre-to-centre distances d between the core subunits alpha 2, beta and beta' presented in the preceding paper, the values of d between initiation factor sigma and alpha 2 (8.4 +/- 1.6 nm), beta (4.4 +/- 2.2 nm) and beta' (10.7 +/- 1.5 nm) were derived from R of sigma (4.1 +/- 0.3 nm) in situ and of the pairs alpha 2--sigma (6.1 +/- 0.4 nm), beta--sigma (5.6 +/- 0.3 nm) and beta'--sigma (7.5 +/- 0.4 nm) within the holoenzyme (alpha 2 beta beta' sigma). The structural parameters of the subunits within their molecular complex are accessible for neutron small-angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of 'hydrogenated' molecular parts and extended exposure times because of weak scattering effects. The overall shape of sigma bound to core enzyme (alpha 2 beta beta') proved to be identical (within experimental resolution) with sigma in the isolated state measured recently by X-ray small-angle scattering. The refined shape of isolated sigma was reduced to an ellipsoid which was orientated with respect to the core structure (alpha 2--beta--beta') in a 'space-filling' way around the position of the sigma centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three-dimensional model.

DNA-Directed RNA Polymerases↗
PubMed1989

Large intramolecular movement in human complement protein C3 induced by methylamine. A small-angle scattering study using monoclonal antibodies as markers.

The reaction of methylamine with complement protein C3, which involves cleavage of a labile thiol ester bond, yields a large intramolecular rearrangement. This is shown by small-angle neutron and X-ray scattering using a Fab antibody as a marker. For the C3(Fab) 1:1 complex, the methylamine reaction yields an increase in the radius of gyration, R, from 4.6 nm to 6.0 nm. In the absence of Fab the corresponding R values increase from 4.4 nm to 5.1 nm. It is estimated that the methylamine-induced increase in R may correspond to a movement of the epitope to a position 5 nm away from the centre of gravity of the C3 molecule. In agreement with this finding, the maximum distance within the C3(Fab) complex increases from 16 nm to 22 nm as a result of the methylamine reaction. In order to explain this conformational change, it is tentatively suggested that the methylamine-induced cleavage of the C3 thiol ester bond leads to a domain rotation within the C3 molecule. In agreement with this idea, the data is consistent with a model which enables a globular domain within the molecule to rotate without redistributing the molecular mass more than that corresponding to the radii of gyration observed.

Antibodies, Monoclonal↗
PubMed1996

Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.

Amides↗
PubMed1997

Neutron crystallographic evidence of lipase-colipase complex activation by a micelle.

The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.

Animals↗
PubMed1997

Molecular dynamics simulation of unsaturated lipid bilayers at low hydration: parameterization and comparison with diffraction studies.

A potential energy function for unsaturated hydrocarbons is proposed and is shown to agree well with experiment, using molecular dynamics simulations of a water/octene interface and a dioleoyl phosphatidylcholine (DOPC) bilayer. The simulation results verify most of the assumptions used in interpreting the DOPC experiments, but suggest a few that should be reconsidered. Comparisons with recent results of a simulation of a dipalmitoyl phosphatidylcholine (DPPC) lipid bilayer show that disorder is comparable, even though the temperature, hydration level, and surface area/lipid for DOPC are lower. These observations highlight the dramatic effects of unsaturation on bilayer structure.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Structural parameters of the Pf1 gene 5 protein-DNA complex in solution by neutron scattering.

Neutron-scattering experiments have been performed on the intracellular complex formed by the gene 5 protein and single-stranded DNA in cells infected by filamentous bacteriophage Pf1. The contrast matched point of the complex (37% 2H2O) is lower than expected and implies that a substantial fraction of potentially labile hydrogen atoms are unable to exchange with the solvent. The mass/length ratio of the complex (3270 daltons/A) indicates an axial subunit repeat of 5.1 A, a value much larger than the subunit repeat previously determined in fibres. The measured value of the cross-sectional radius of gyration at infinite contrast (Rc = 43.3 A) indicates an outer radius of 60 to 63 A for the complex. The variation in Rc with contrast shows that regions of higher scattering density are located, on average, towards the outside of the complex. The high-angle region of the intensity curve (measured in 2H2O) reveals a clear subsidiary maximum at 0.105 A-1 arising from the 60 A helical pitch of the nucleoprotein complex. The structural parameters of the Pf1 gene 5 protein-DNA complex in solution are compared with those of the fd gene 5 protein-DNA complex.

Bacteriophages↗
PubMed1982

Solution scattering studies of dimeric and tetrameric spectrin.

The structure of spectrin dimers and tetramers in solution has been examined by light, low-angle X-ray and neutron scattering. The results show a good correspondence between the solution dimensions of these molecules and their appearance in the electron microscope after shadowing. The scattering profiles are not compatible with an extended rod-like character, but reflect the presence of a considerable degree of bending. The radii of gyration of the dimer and tetramer were determined to be 170 and 375 A and the cross-section radii of gyration 14 and 12.3 A, respectively. Both are thus long, thin, rather bent molecules, and the tetramer is twice the length of the dimer.

Humans↗
PubMed2000

Structural models of lipid surface monolayers from X-ray and neutron reflectivity measurements.

Structural investigations of phospholipid monolayers on aqueous subphases on the submolecular level using X-ray and neutron reflectivity measurements are reviewed. While such investigations have been limited in the past by a relatively restricted accessible momentum transfer range, recent developments in synchrotron technology--almost doubling this range--have considerably improved the capabilities of the technique. Until recently, data interpretation has entirely relied on 'box models' which describe the structures as molecularly homogeneous slabs--one hydrophobic and one hydrophilic. It is shown that box models of phospholipid monolayers are rather inadequate to model data at the high momentum transfer available nowadays in X-ray measurements. As an alternative, a hybrid data inversion strategy is proposed that treats the hydrophobic alkane phase as a homogeneous slab and describes the position of submolecular fragments of the lipid headgroups by means of distribution functions along the interface. Within this approach, composition-space refinement--enabling the coupling of data sets from various X-ray and neutron contrasts--in connection with volumetric constraints enables structural characterization of lipid monolayers in unprecedented detail. Extending a recent characterization of dimyristoylphosphatidic acid (DMPA) monolayers on pure water [Schalke et al., Biochim. Biophys. Acta 1464 (2000) 113-126] it is shown that stoichiometric binding of the divalent cations--DMPA-:Cat2+= 2:1--occurs only at exceedingly low areas per molecule, A lipid. At low surface pressure pi, both cations and anions are incorporated into the headgroup in significant amounts, approximately 0.68 Ba2+ and approximately 0.35 Cl- per PA molecule at pi = 2 mN m(-1). They are continuously squeezed out upon compression, until upon approaching Alipid = 41 A2, the stoichiometric ratio between bound cations and acidic headgroups is observed. The average inclination angle alpha of the headgroups as well as their water content is constant along the whole isotherm. The intrinsic contribution to the distribution width--i.e. the spread that is due to a distribution of the fragments within the headgroup without the action of capillary waves--increases with compression up to pi approximately 30 mN m(-1) and drops sharply thereafter in a regime of the isotherm where Alipid approaches its limiting value. The same general picture is observed for DMPA on subphases with 10 mM Ca2+, although the lower electron density of that cation limits the precision of the results.

Aniline Compounds↗
PubMed2005

Protein crystal perfection and its application.

Knowledge of protein crystal perfection in theory and practice is reviewed. X-ray methods of assessing perfection such as mosaicity, topography and reciprocal-space mapping are described. X-ray diffraction physics applications of protein crystals such as in Laue geometry, the large-angle oscillation technique and forming polychromatic profiles across diffraction spots are covered. Cryo- and room-temperature cases are discussed including in X-ray and neutron protein crystallography. Experience of freezing very large crystals, which are commonly used today in neutron protein crystallography, is highlighted.

Crystallization↗
PubMed2001

Porous calcium polyphosphate scaffolds for bone substitute applications -- in vitro characterization.

Porous structures were formed by gravity sintering calcium polyphosphate (CPP) particles of either 106-150 or 150-250 microm size to form samples with 30-45 vol% porosity with pore sizes in the range of 100 microm (40-140 microm). Tensile strength of the samples assessed by diametral compression testing indicated relatively high values for porous ceramics with a maximum strength of 24.1 MPa for samples made using the finer particles (106-150 microm). X-ray diffraction studies of the sintered samples indicated the formation of beta-CPP from the starting amorphous powders. In vitro aging in 0.1 M tris-buffered solution (pH 7.4) or 0.05 M potassium hydrogen phthalate buffered solution (pH 4.0) at 37 degreesC for periods up to 30d indicated an initial rapid loss of strength and P elution by 1 d followed by a more gradual continuing strength and P loss resulting in strengths at 30d equal to about one-third the initial value. The observed structures, strengths and in vitro degradation characteristics of the porous CPP samples suggested their potential usefulness as bone substitute materials pending subsequent in vivo behaviour assessment.

Bone Substitutes↗
PubMed1982

Diet and the evolution of modern human form in the Middle East.

Fully modern human form more gracile than the antecedent archaic modern form was evident by 30,000 years ago. One hypothesis to explain this decrease in skeletal robustness is that change occurred in human diet and that this change was associated with a decrease in activity levels required in both individual and group behavior. It is possible to study dietary change directly using trace element analysis of strontium levels in bone. The amount of strontium in bone reflects the amount of strontium in diet. Since plants contain higher levels of strontium than do animal soft tissues, the level of bone strontium will differ between individuals according to the proportion of plant and animal products in their diets. In this study the ratio of strontium:calcium in human bone to strontium:calcium in faunal bone is compared for samples of archaic modern humans (from Mugharet et Tabun, Mugharet es-Skhul, and Jebel Qafzeh) and fully modrn humans from Mugharet el-Kebara and Mugharet el-Wad) from Israel. The use of a ratio controls for potentially unequal strontium levels in soils at different sites and for different diagenetic histories between sites. The results of the analysis are internally reliable, reflecting bone strontium levels rather than technique error; therefore, they reflect diet. It appears that a change occurred in the amount of animal protein in the diet of humans but that this change occurred almost 20,000 years after the first appearance of skeletally modern humans. These results refute the hypothesis that the morphological transformation to modern human form occurred as a result of behavioral changes involved in obtaining previously unused foods. If any decrease in human activity level occurred between archaic modern and fully modern humans, this decrease probably was due to alterations in the means of procuring or processing the same kinds of foods that had been utilized earlier in time.

Animals↗