Small-angle X-ray scattering of human serum high-density lipoproteins

Small-angle x-ray scattering (SAXRS) studies of the human serum high-density lipoprotein HDL₂ indicate a symmetrical particle with a radius of gyration Rg = 46 Å. The positions and intensities of subsidiary maxima in the scattering curves are not consistent with those of a uniformly electron dense sphere. Scattering curves calculated for spheres with a step-model radial electron density distribution, show good agreement with the experimental scattering curve for HDL₂ only for specific values of the step function used. The dimensions obtained for the electron-deficient core and electron-rich shell model are quantitatively consistent with a predominantly surface location for the HDL₂ protein and phospholipid head groups, the more hydrocarbon species being located in the interior of the particle.     

Small-angle X-ray scattering of D-Ribulose-1,5-diphosphate carboxylase from Dasycladus clavaeformis roth (Ag.) in solution

D-Ribulose-1,5-diphosphate carboxylase from $\text{Dasycladus}$ was purified, and the gross dimensions were obtained by means of small-angle X-ray scattering measurements in solution. Dissolved single crystals of this enzyme (called “fraction I protein”) gave the same hydrodynamic parameters as the purified form. The molecular weight was found to be 535,000, and a radius of gyration of R_g = 45.5 Å was determined. The experimental scattering curves revealed a geometrical particle of D-Ribulose-1,5-diphosphate carboxylase with gross dimensions of that of a hollow sphere with outer radius of 56 Å and inner radius of 12 Å. Determinations of the diffusion coefficients lead to the conclusion that the enzyme has a spherical shape of almost uniform density.     

On the subunit structure of crotoxin: hydrodynamic and shape properties of crotoxin, phospholipase A and crotapotin.

This paper reports physical-chemical properties of the subunit structure of crotoxin, phospholipase A and crotapotin. The native crotoxin has a sedimentation coefficient of 3S and a radius of gyration of R_g = 16.5 Å and a molecular weight of 30,900. Dissociation of the 3S particle results in two proteins of unequal size with sedimentation coefficients of 1.5 S (crotapotin) and 1S (phospholipase A). These dissociated species and the reconstituted complex were investigated by means of hydrodynamic methods including small angle X-ray scattering. The actual frictional ratios were obtained indicating that crotoxin is a sphere with a Stokes' radius of R_o = 22.5 Å and an axial ratio of 1:3, whereas phospholipase A, depending on the degree of association, has a radius of gyration of R_g = 32.4 Å and a high axial ratio of 1:14 for the monomer. Crotapotin has a radius of gyration of R_g = 12.4 Å, indicating an oblate ellipsoid of revolution of an axial ratio of 1:4. Evidently, the crotoxin complex consists of one highly asymmetric molecule (phospholipase A) and an oblate ellipsoid (crotapotin), which reconstitutes to a spherical 3S-particle (crotoxin).     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

An x-ray scattering investigation of broad bean mottle virus in solutions of various electron densities

Low-angle X-ray scattering data to a resolution of 30 Å are presented for broad bean mottle virus suspended in buffer and in solutions of higher electron density produced by the addition of sucrose or the trisaccharide melezitose. Comparison of the scattered intensity distributions with those of simple model particles are made and radial electron density distributions are obtained. The results indicate that in buffer the virus particle has a radius of gyration of 117 Å, a mean outer radius of about 147 Å, and a nearly hollow core of about 60 Å radius. The scattering data for the virus in sugar solutions supports these results and indicates that much of the region within the virus open to water is also open to penetration by the sugar molecules. Melezitose can penetrate about 60% of the volume of the virus open to water while sucrose can penetrate nearly 90%. The region of the virus within 90 Å from the center is more easily penetrated by these sugars than the region from 90 Å to the surface. It is concluded that the virus at this resolution appears as a hollow, approximately spherically symmetric object with a high density and probably well organized RNA region enclosed by a protein shell into which some of the RNA penetrates.     

Shape and size of bovine rhodopsin: A small-angle X-ray scattering study of a rhodopsin-detergent complex

Rhodopsin is extracted from rod outer segments of retinas with dodecyldimethylamine oxide (DDAO), a non-ionie detergent. The rhodopsin-DDAO complex is characterized by binding experiments, gel filtration, sedimentation, densimetry; its homogeneity, chemical composition, weight and partial specific volume are determined. The complex turns out to be a reasonably monodisperse association of one rhodopsin and 156 DDAO molecules. The rhodopsin-DDAO complex and the detergent micelles are studied by small-angle X-ray scattering techniques using a water/sucrose solvent of variable density. The experiments are performed on an absolute scale; mainly the value and curvature of the scattering curves at zero angle are exploited. The structure of the complex and of the micelles is shown to be independent of sucrose. Under these conditions the final result of the X-ray scattering study of each type of particle is the numerical value of a set of five parameters: molecular weight, volume and radius of gyration of the volume occupied by the particles, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of rhodopsin and of the detergent moiety are very near to each other. The analysis of these parameters leads to the determination of the size and shape of the detergent micelles and to an estimate of the size and shape of the volumes occupied by protein and by detergent in the complex. We find rhodopsin to be a very elongated molecule (maximum diameter ~95 Å) which spans a flat detergent micelle. These results suggest that in the rod outer segment discs the rhodopsin molecules span the membranes, that the rhodopsin molecules of the two opposite membranes of each disc come near to each other and that a high fraction of the intra-disc space is occupied by rhodopsin.     

Low-angle neutron scattering from chromatin subunit particles.

Monomer chromatin particles containing 140 base pairs of DNA and eight histone molecules have been studied by neutron scattering. From measurements in various H2O/D2O mixtures, radii of gyration and the average scattering density of the particle were determined. The radius of gyration under conditions when scattering from the DNA dominates is 50A, and when scattering from the protein dominates, 30A. Consequently the core of the particle is largely occupied by the histones while the outer shell consists of DNA together with some of the histone.     

Small-angle X-ray scattering studies of Escherichia colil-asparaginase

Small angle X-ray scattering studies on Escherichia colil-asparaginase solutions show that the enzyme has a radius of gyration of 34.0 Å ± 0.5 Å at pH 7. The radius of gyration of the dissociated monomer is 16.0 Å ± 1.0 Å; it has the general shape of a prolate ellipsoid with an axial ratio of 1.4. A tetramer of four such ellipsoids arranged with 222 symmetry gives good agreement between measured and calculated radii of gyration if the distance between subunit centers is 43 Å. The tetramer dissociates on dilution below 1% and at pH values below 3.0. Acid-induced denaturation at pH 2.0 is irreversible in contrast to the reversible guanidine-HCl-induced denaturation.     

HDL Subspecies in Young Adult Twins: Heritability and Impact of Overweight

The association between abdominal obesity and atherogenic lipid profile emerges from complex interactions of genes and environment. We aimed to explore the heritability and effects of overweight on serum lipid profile (high‐density lipoprotein‐cholesterol (HDL‐C), HDL mean particle size, percentages of HDL_{2b, 2a, 3a, 3b, and 3c}, low‐density lipoprotein‐cholesterol (LDL‐C), LDL peak particle size and triglycerides (TGs)) in healthy, young adults. HDL‐C, LDL‐C, and TG were measured in 52 monozygotic (MZ) and 89 dizygotic (DZ) twin pairs, aged 23–32 years, chosen to represent a wide range of BMIs (17.6–42.9 kg/m²). Of them, 24 MZ and 26 DZ pairs were chosen at random for measurements of HDL mean and LDL peak particle sizes and percentages of HDL subspecies. The heritabilities of the lipid parameters adjusted for BMI were HDL‐C 73%, HDL mean particle size 56%, HDL subspecies 46–63%, LDL‐C 79%, LDL peak particle size 49%, and TG 64%. Genetic and environmental correlations between BMI and HDL‐C, LDL‐C, and TG were modest (0.3–0.4). Abdominal overweight (waist circumference ≥94 cm for males and ≥80 cm for females) associated with decreased HDL‐C, increased LDL‐C, and TG concentrations, smaller HDL mean particle size, lower HDL_2b, and higher HDL_3c percentages in both genders. Within MZ twins, controlling for genetic influences, within‐pair differences in HDL_3c percentage were associated with those in waist (r = 0.46, P = 0.032) and BMI (r = 0.51, P = 0.013). In conclusion, serum lipid parameters, including LDL peak and HDL mean particle sizes and HDL subspecies distribution are under strong genetic control. Overweight associated with significant lipid profile changes, particularly, small HDL_3c increased in overweight independent of genetic influences.     

On the quaternary structure of native rabbit muscle phosphofructokinase.

Small angle X-ray scattering measurements on solutions of native rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP; D-fructose-6-phosphate 1 phosphotransferase) show that the dimer has a radius of gyration of 32.5 Å and a molecular weight of 160,000, and that the biologically active tetramer has a radius of gyration of 51.5 Å and a molecular weight of 320.000. A possible model was calculated from scattering curves of the dimer and tetramer suggesting two hollow cylinders with cell dimensions for the dimer of a height of 78.0 Å and a long half axis of 38.0 Å, and for the tetramer of a height of 155.0 Å and an outer radius of 35.0 Å. The tetramer is formed along the 78.0 Å axis of the dimer by means of an end-to-end aggregation. The overall particle dimensions of the protomer of molecular weight 80,000 is calculated to be 35.0 × 30.0 × 55.0 Å, assuming an elliptical molecule. The distance between the centers of the two dimeric units within the tetramer is 104.5 ± 1.5 Å.     

Lipoprotein geometry. II. Apoprotein exchange in human plasma high density lipoprotein.

The relationships between the apoproteins of intact human serum high density lipoprotein particles, HDL₂ and HDL₃, have been studied by observing the exchange of radioactively labeled apoproteins between one subclass and the other. This exchange process can be inhibited by chemically crosslinking the apoproteins of either the labeled or unlabeled subclass. These results are consistent with a dynamic relationship between HDL₂ and HDL₃ which appears dependent upon the association and perhaps the conformation of the apoprotein components of the lipoprotein particles.     

Physicochemical characterization of soluble complexes of human serum low density lipoprotein with heparin

Soluble complexes of low density lipoproteins (LDL) with heparin (HEP) and chondroitin sulphate (CS) in the absence of divalent cations have been studied by means of a micro-rolling-ball ciscometer to obtain information about molecular size and structure of the aggregates. The rheological results were supplied and corroborated by light scattering measurements, electrophoresis and analytical ultracentrifugation. Molar binding ratios were measured using gel filtration assays and ultracentrifugation. At a certain weight ratio of LD To HEP the solutions showed a significant viscosity maximum. At this weight ratio 2–3 LDL particles are held together 1–2 HEP chains. The hydrodynamic radius R_H of this complex is about 16.3 ± 0.90 nm and the rotational diffusion constant is > 7.1 × 10³ s^?1. With excess HEP the radius of the aggregates is almost the same as that of free LDL (R_H = 11.9 ± 0.70 nm). Quantitative binding studies revealed that in this case 1–2 HEP molecules are bound to a single LDL particle. An interaction was also found with CS and LDL but complex formation in this case showed different characteristics. Very low density lipoproteins (VLDL) and high density lipoproteins (HDL) gave no rheologically effective aggregates with HEP.     

Studies on deoxyribonucleoprotein structure. Small-angle X-ray scattering in solutions of various ionic strengths.

The cross-sectional radius of gyration of the deoxyribonucleoprotein (DNP) threads was measured by small-angle X-ray scattering in a wide range of ionic strengths (from 0.0005 to 2 M NaCl). For DNP in a solution of low ionic strength, this value is 30 Å. The increase of ionic strength results in partial deproteinization of DNP, while the cross-sectional radius of gyration varies from 25 Å for DNP in 0.7 M NaCl to 10 Å for DNP in 2 M NaCl. It is suggested that gradual deproteinization by the increase of NaCl concentration causes conformational changes, which are associated with the alteration of the DNP superstructure. The data are interpreted on the basis of the superhelical model of DNA packing in DNP; however, the coexistence of superhelical and unfolded regions in the DNP structure is also a possibility.     

Particle size interconversion of human low density lipoproteins during incubation of plasma with phosphatidylcholine vesicles

Incubation of plasma (37°C, 6hr) in the presence of increasing amounts of phosphatidylcholine (PC) vesicles, above a threshold concentration, results in an increase in particle diameter of LDL relative to that from nonincubated plasma. With further PC addition, the major peak of LDL in the gradient gel electrophoretic pattern is transformed, first, into a bimodal and, subsequently, into a single peak distribution. PC-induced interconversion of LDL requires factors(s) in the d > 1.20 g/ml fraction and, at PC concentrations below approximately 2 mg/ml, is not inhibited by p-chloromercuriphenylsulfonic acid. Plasma incubation with increasing PC levels also leads to characteristic particle size transformations in HDL₃ species, with the transformation products ultimately converging to form a single peak pattern within the HDL_2a size interval. In certain subjects, incubation of plasma, in the absence of added PC, shifts the particle size distribution of LDL towards smaller species; this can be prevented by addition of PC. We propose that incubation-induced shifts of LDL towards larger or smaller species result from changes in phospholipid (PL) content of LDL.     

Neutron small angle scattering of the Mo-Fe protein (nitrogenase) from Clostridium pasteurianum

Neutron small angle scattering measurements of solutions of the Mo-Fe protein from $\text{C. pasteurianum}$ have yielded the following results. The molecular weight of the protein is 208,000 ± 10,000, in agreement with figures obtained by other methods. The radius of gyration is 39.8 ± 0.7 Å in H₂O, and 37.6 ± 0.3 Å in D₂O. The experimental scattering curves have been compared with the calculated scattering curves of simple homogeneous bodies. It is concluded that the MoFe protein from $\text{C. pasteurianum}$ is a non spherical particle having an axial ratio of 2:1, and that it probably has little, if any, solvent containing cavities.     

Analysis of local order in the spatial distribution of cell surface molecular assemblies.

Electron micrographs obtained from platinum-carbon replicas of freeze-cleaved or freeze-dried cells and exhibiting intramembranous or externally-disposed particles were analysed statistically for degree of non-random particle association. Analysis was based on use of an analogy to the radial distribution function, g(r), which provided the average density of neighbor particles around a particle in the micrograph at 30 Å increments from particle center. The ratio of average density of neighbors around a particle to overall particle density in the micrograph was then determined as a function of distance from particle center. For a random plot of 90 Å particles, the computed ratio deviated from 1 by less than 10% for a distance up to 900 Å from particle center. In a field of similarly-sized intramembranous particles within the freeze-cleaved surface membrane of a mouse fibroblast, there were indications of slight non-random particle associations at distances of 120–240 Å and 300–330 Å from particle center, while a significant association of particles comprising an intercellular gap junction was obtained at 90–120 Å. Virus-related molecular assemblies on the surfaces of fibroblasts infected with a temperature-sensitive mutant of murine luekemia virus and grown under conditions that did not permit virus assembly to occur were analysed similarly. Significant deviation from randomness was found at several distances from particle center, both on areas of membrane that were populated by large numbers of particles and on low-density areas.     

Small-angle X-ray scattering studies of the structure of nucleosome core histone complexes in solution

The nucleosome core histone complex in solution at 2 M NaCl and pH 7 has a radius of gyration R_s, of 3.48 nm and a maximum dimension, L, of 12 nm. Its shape is disc-like with a mean thickness of 3 nm. The radius of gyration determined by us is of the same value as the radius of gyration of the complex in intact core particles (Braddock) et al., Biopolymers 1981, 20, 327). Thus, we conclude that the basic histone tails of the protein complex project about 2 nm from its central part.