Phytohemagglutinin and transmembrane proteins in agglutinated sheep erythrocyte ghost membranes

Oriented and periodically stacked sheep erythrocyte ghost membrane specimens were prepared by agglutination of the ghosts with phytohemagglutinin M and sedimentation, and were studied by X-ray diffraction. The spatial orientation of the planes of the membranes in the diffracting stack was determined from the lamellar reflections of the periodic stacking. Equatorial diffraction at (10.5 Å)⁻¹ and a (1.5 Å)⁻¹ reflection were recorded which correlate with side-to-side packed transmembrane α-helices in the agglutinated membrane. A broad (4.6 Å)⁻¹ ring with strong equatorial accentuation and broad maxima at about (2.2 Å)⁻¹ and (1.2 Å)⁻¹ were observed which are attributed to the hydrocarbon chain arrangement in lipid phases of the agglutinated ghost membrane.     

Studies of a serum albumin-liposome complex as a model lipoprotein membrane

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicular, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associate with lipid.Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 Å. The same lamellar separation in the positively charged system is 108 Å. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 Å while that for the positively charged lipoprotein system is 91 Å. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer.Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 Å spacing, characteristic of hexagonal packing of the hydrocarbon chains.A classical light scattering technique is to used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose < arabinose < malonamide < glycerol). K⁺ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

Interaction of amphotericin B with monolayers of egg lecithin and cholesterol: polorized absorption spectra

Polarized absorption spectra have been obtained of the antibiotic polyene, amphotericin B, interacting with monolayers of egg lecithin, cholesterol and equimolar egg lecithin-cholesterol at low and high surface pressures. An expression is derived which enables the determination from the polarization data of the orientations of the transition moments of the polyene absorption bands at 4077 and 3645 Å. For some of the systems the 3645-Å band is replaced by a previously unreported band appearing between 3610 and 3632 Å. The orientation of the 4077-Å transition moment (parallel to the long molecular axis) is found to vary from an angle of 64 ° with the surface for the low-pressure monolayers of cholesterol to 21 ° for the high-pressure films of egg lecithin-cholesterol. For the band between 3610 Å and 3645 Å, the angle varies from 90 ° for cholesterol to 18 ° for the high-pressure mixed-lipid film. It is found that a large increase in surface pressure of the cholesterol and egg lecithin-cholesterol monolayers causes a decrease in the angle of the 4077-Å moment for both films and that of the higher energy moment for the mixed film. Increasing the content of cholesterol in these monolayers rotates the orientation of the transition moments for both bands toward the surface normal, the change being greatest for the low-pressure films. The effectiveness of amphotericin B in lowering the surface tension of these lipid monolayers is related to its binding, orientation and extent of penetration. For low-pressure cholesterol films where the surface interaction with the polyene is greatest, the binding and penetration are large and the polyene molecule is oriented with its long dimension nearly perpendicular to the surface.     

Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

New Crystal Forms of the Motile Major Sperm Protein (MSP) ofAscaris suum

We have obtained two new crystal forms of theAscarismajor sperm protein (MSP) that mediates amoeboid cell motility in nematode sperm. We obtained crystals with C2 symmetry from bacterially expressed α-MSP witha= 216.5 Å,b= 38.6 Å,c= 32.5 Å, γ = 93.1° and also crystals with P2₁symmetry from native β-MSP witha= 63.1 Å,b= 91.7 Å,c= 72.5 Å, γ = 91.3°. A full native data set has been collected for each crystal form using synchrotron radiation. Both crystal forms diffract to 2 Å and are suitable for high-resolution structural investigation.     

A Small Angle X-Ray Scattering Study of the Binding of Cyclosporin-A to Calmodulin

Small angle X-ray scattering (SAXS) was applied to the binding of the immunosuppressant drug cyclasporin-A to the protein calmodulin. Guinier analysis of the SAXS profiles yielded a radius of gyration, Rg, of 19.7 ± 0.3 Å for the native protein and 16.9 ± 0.3 Å for the drug/protein complex. Maximum entropy (maxent) methods of data analysis were used to calculate the distance distribution function, p(r). From this analysis, the R_g for the native protein is 20.9 ± 0.1 Å and that for the complex 16.7 ± 0.1 Å. The measured SAXS profiles and the derived p(r) for calmodulin agree with profiles calculated from the crystallographic structure of calmodulin. Major structural changes are induced in calmodulin on binding cyclosporin-A. A model consistent with the observed scattering profiles is an ellipsoid with major axes 55 and 36 Å. Molecular modeling of the calmodulin molecule suggests that bond rotation in the flexible α-helix linker region produces models consistent with the above observations.     

Crystallization and Preliminary X-Ray Analysis of Borrelia burgdorferi Outer Surface Protein A (OspA) Complexed with a Murine Monoclonal Antibody Fab Fragment

The Borrelia burgdorferi outer surface lipoprotein OspA is a current focus for vaccine development to prevent Lyme disease infection. A soluble, recombinant form of the protein lacking the aminoterminal lipid membrane anchor was cocrystallized with the Fab fragment of an agglutinating mouse monoclonal antibody. The crystals belong to space group P2₁2₁2₁, with a = 90.0 Å, b = 91.9 Å, and c = 102.9 Å and they were found to diffract to a maximum resolution of 2.8 Å using synchrotron radiation.     

Studies on the role of histones H1 (f1) and H5 (f2c) in chromatin structure

Chicken erythrocyte and liver nuclei, isolated and fixed in isotonic saline, contained compact chromatin fibers about 200 Å in diameter. Fibers very similar in dimension and appearance are usually visible in thin sections of fixed nuclei in situ and probably represent chromatin organization close to the native state. After suspension of isolated nuclei in a mildly hypotonic buffer, chromatin fibers extended, became reduced in diameter and apparently unraveled in places. Under such conditions, new detail was revealed suggestive of both helical structure and of subunit organization. The fibers extended and contracted reversibly, changing in diameter from 200 Å to about 100 Å when alternately exposed to isotonic saline and to distilled water. The 200 Å fibers were irreversibly lost, however, following extraction of nuclei with high concentrations of NaCl which selectively removed H1 from liver and H1 plus H5 from erythrocyte chromatin. After extraction of more tightly complexed histones, the residual chromatin consisted mainly of fine filaments less than 30 Å thick. These results suggest a model for native chromatin fibers in which sub-unit organization and coiled configuration are combined, and in which histones H1 and H5 play an integral role in the maintenance of structure.     

Crystallization and Preliminary X-Ray Diffraction Analysis of the 190-Å-Long Coiled-Coil Dimerization Domain of the Actin-Bundling Protein Cortexillin I fromDictyostelium discoideum

We have crystallized the ≈190-Å-long parallel two-stranded coiled-coil oligomerization domain of the actin-bundling protein cortexillin I fromDictyostelium discoideum. The orthorhombic crystals belong to the space group C222₁with unit cell dimensions ofa= 71.3 Å,b= 127.8 Å, andc= 91.6 Å. As both native and selenomethionine-substituted protein crystals diffract to 3.0 and 2.85 Å resolution, respectively, using synchrotron radiation, they are suitable for the first high-resolution structural analysis of a two-stranded coiled coil comprising more than six heptad repeats. Moreover, because the polypeptide chain fragment contains a recently identified two-heptad-repeat long sequence that is indispensable for the assembly of the cortexillin I coiled-coil oligomerization domain, its high-resolution structure should enable us to extend our knowledge on the molecular mechanisms underlaying coiled-coil formation and to establish the precise manner in which the two “trigger” sequences interact with one another in the dimer.     

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage φ29 connector particle

The connector or portal particle from double-stranded DNA bacteriophage φ29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a=416.86 Å, b=227.62 Å, c=236.68 Å and β=96.3° and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4₂2₁2 with cell dimensions a=b=170.2 Å, c=156.9 Å and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 Å respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.     

Crystallization and Preliminary X-Ray Analysis of Thermoactinomyces vulgaris R-47 α-Amylase II

Crystals of Thermoactinomyces vulgaris α-amylase II, which is a new type of α-amylase having hydrolysis activities for pullulan and cyclodextrins, have been obtained and diffraction data to 2.9 Å resolution were collected. The crystal belongs to an orthorhombic system with cell dimensions of a = 119.5 Å, b = 120.6 Å, and c = 114.6 Å and a space group of P 2₁2₁2₁. Two or three protein molecules are expected to exist in an asymmetric unit.     

Translocation of hyaluronic acid in cell surface of cultured mammalian cells after X-irradiation and its recovery by added adenosine triphosphate

To investigate the mechanism of radiation-induced decrease in cell electrophoretic mobility and its recovery by added adenosine triphosphate, specific enzymes and buffer solutions of different ionic strength were utilized. Decrease in the mobility of irradiated cells was detected only with the buffer solution of ionic strengths higher than 0.100. In this range of ionic strengths, removal of hyaluronic acid from cell surface by hyaluronidase had no effect on the electrophoretic mobility of irradiated cells, while the enzyme treatment resulted in 27% mobility reduction in non-irradiated cells. The removal of sialic acid and chondroitin sulfate by their specific enzymes resulted in the similar decrease in mobility either in irradiated and non-irradiated cells. These results suggest that the X-ray induced translocation of hyaluronic acid from the peripheral zone of 0–7.5 Å into the deeper zone of about 10–17 Å, if we use the Debye-Hückel's thickness of ion atmosphere for an approximate estimate of effective depth of electrokinetic plane of shear. Hyaluronic acid reappeared to the peripheral zone by the subsequent incubation after small dose irradiation, or by the addition of 1 mM adenosine triphosphate with Ca²⁺.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : V. THE INFLUENCE OF THE THICKNESS OF DRIED COLLODION MEMBRANES UPON THEIR ELECTROMOTIVE BEHAVIOR

1. Experiments were carried out to decide whether or not the electromotive properties of dried collodion membranes depend upon their thickness. 2. A number of dried collodion membranes of varying thickness, 3–160 µ, were prepared from collodion preparations of different electrochemical activity. The characteristic concentration potentials across them were measured and the means of these values determined for each thickness. 3. The characteristic concentration potentials across dried collodion membranes are a function of their thickness. The thinnest membranes yield in all cases the lowest concentration potentials; increasingly thicker membranes give increasingly higher potential values, until a constant value is reached which is characteristic of the particular collodion preparation used. With electrochemically active collodion, characteristic concentration potentials approaching the thermodynamically possible maximum are obtained with membranes of only 10 µ thickness, thinner membranes giving appreciably lower values. With two rather inactive commercial collodion preparations the characteristic concentration potential increases from about 30 mv. for membranes 3 µ thick to about 42 mv. for 20 µ membranes; still thicker membranes do not show a significant increase in the potential values. With a highly purified collodion preparation the constant maximum value was found to be about 32 mv., 4 µ thick membranes giving only about 22 mv. 4. These results do not support the homogeneous phase theory as applied to the dried collodion membrane. They are readily compatible with the micellar-structural theory. Several special possible cases of the latter as applied to the dried collodion membrane are discussed.     

Small liposomes are better than large liposomes for specific drug delivery in vitro

We have compared drug transfer into target cells in vitro from liposomes of different sizes. Liposomes of mean diameter 800 Å, 2000 Å or 4000 Å, containing the folate analogue, methotrexate, and the fluorophore, carboxyfluorescein, were covalently coupled to Staphylococcus aureus protein A. Cells of the murine k haplotype were preincubated with an anti-H-2K^k monoclonal antibody. Excess antibody was removed and then cells were incubated with liposomes. The number of cell-bound liposomes was determined by fluorimetry. The drug effect was assayed by the methotrexate-mediated inhibition of radiolabeled deoxyuridine uptake. The drug effect was more important in the case of the 800 Å vesicles than for the larger liposomes, despite the fact that the quantity of drug bound to cells was several-fold greater for large liposomes than for small ones. Since fusion is excluded by the non-proportionality of drug binding and drug effect, the predominant manner of liposome entry seems to be endocytosis. At least for these in vitro studies, the endocytosis by target cells of small liposomes seems to be more efficient than that of large liposomes.     

Activation of recA protein: The salt-induced structural transition

Purified recA protein is induced by high salt concentrations to hydrolyse ATP even in the absence of DNA. By small angle neutron scattering we show that this salt activation results from a structural transition of the protein filament in the presence of ATPγS from the inactive, compact form (a helical polymer of pitch 70 Å and cross-sectional radius of gyration R_c 40 Å) to the open form (a helical filament of pitch 95 Å and R_c 35 Å, which are the same structural parameters as in the ATPase active complex with DNA and ATP), without detectable change in the degree of association. We conclude that activation of recA is due to the same structural change whether induced by the binding of DNA or by salt. Indeed, the other enzymatic activity of recA, the proteolytic cleavage of the lexA repressor, is found to be inducible by the same salt concentrations as those of the structural transition.     

Three-Dimensional Structure of the Porcine Gastric H,K-ATPase from Negatively Stained Crystals

A low-resolution three-dimensional model of membrane-bound H,K-ATPase from pig gastric mucosa has been reconstructed by electron microscopy and image processing of two-dimensional crystals in negative stain. The crystal formation is induced by magnesium and vanadate, which stabilize the E₂conformation of the enzyme. The unit cell, with a size ofa=b= 123 Å, γ = 90°, has tetragonal p4 symmetry. There are four separate αβ protomers within each unit cell. The high-contrast region is limited to the cytoplasmic part of the protein. The total volume of the observed asymmetric protein domain corresponds to a molecular mass of 80–90 kDa. It consists mainly of a large pear-shaped domain measuring 60 × 45 Ų, with a height of 50 Å as measured perpendicular to the membrane plane. A small stalk segment, 20 Å in length, forms a connection to the transmembrane region.     

The structure of oriented sphingomyelin bilayers

X-ray diffraction from oriented bilayers of sphingomyelin gave up to 14 orders of diffraction of a lamellar repeat of 68.5 Å on the meridian and up to eight reflections, including a strong reflection at 4.2 Å, on the equator. The diffraction spacings did not change when the sphingomyelin bilayers were exposed to different humidities. A direct analysis of the low resolution X-ray data, using deconvolution is presented. A comparison of the Patterson functions of sphingomyelin with those of phosphatidylcholine and phosphatidylethanolamine suggests that the molecular structure of sphingomyelin in oriented bilayers resembles the structure of both phosphatidylcholine and phosphatidylethanolamine. Molecular model calculations for sphingomyelin bilayers have also been performed. Electron density profiles of sphingomyelin bilayers at resolution of about 6 Å and about 2.5 Å are presented. Our results indicate that the phosphorylcholine head group of sphingomyelin is in the plane of the membrane and at right angles to the hydrocarbon chains, the hydrocarbon chains are nearly parallel to each other, and there is only a limited, if any, interdigitation of the hydrocarbon chains of the adjacent sphingomyelin molecules in the bilayer.     

Crystallization and Preliminary X-Ray Structure Analysis of 1-Aminocyclopropane-1-carboxylic Acid Deaminase

The enzyme 1-aminocyclopropane-1-carboxylic acid deaminase from the bacterium Pseudomonas sp. has been crystallized using the hanging-drop method. The crystals belong to the orthorhombic space group P 2₁2₁2₁ (a = 70.0 Å, b = 70.0 Å, c = 355.0 Å). An asymmetrical unit contains two trimer molecules of M_r = 110,000. The diffraction data have been collected to 3.5-Å resolution. Analysis of the data using the self-rotation function suggests threefold axes within the trimer molecules and a pseudotetragonal arrangement between the trimer molecules in the cell.     

Study of water permeability through phospholipid vesicle membranes by O NMR

Vesicle suspensions of up to 5 % egg lecithin and 2.5 % cholesterol have been found to have no effect on the NMR relaxation times of ¹⁷O from water. Addition of 1–5 mM Mn²⁺ to an equimolar vesicle suspension of egg lecithin and cholesterol permitted resolution of the free induction decay into two exponential components, a fast one arising from the external water and a slow one arising from the intravesicular fluid. From the rates of relaxation the mean life time of the water molecules within the vesicles was calculated to be 1±0.1 ms at 22°C. The size of the vesicle was estimated from electron micrographs to be about 500 Å in diameter. These data yield an equilibrium water permeability, P_w, of about 8 μs⁻¹ for the vesicle membranes. From the temperature dependence of P_w an activation energy of 12±2 kcal/mol was obtained. The longitudinal relaxation time (T₁) of water within vesicles remained the same as in pure water.     

Synthesis and structure of an organosamarium aryloxide complex, (C5 Me5)2 Sm(OC6HMe4-2,3,5,6)

Bis(pentamethylcyclopentadienyl)samarium bis- (tetrahydrofuranate), (C₅Me₅)₂Sm(THF)₂, reacts with 2,3,5,6-tetramethylphenol in toluene to yield (C₅Me₅)₂Sm(OC₆HMe₄-2,3,5,6). The compound crystallizes in the space group P2₁/c with a = 8.725(3) Å, b=18.821(6) Å, c=18.461(6) Å, β= 111.17(2)°, V = 2827(2) ų and D_c=1.340 g cm⁻³ for Z = 4. Molecules of the aryloxide complex are monomeric and exhibit a bent metallocene structure with a nearly linear Sm---O---C(aryloxide) linkage: Sm---O = 2.13(1) Å, O---C = 1.29(2) Å, and Sm---O---C = 172.3(13)°. Reaction of the samarium complex with phenyllithium produces the previously- characterized species (C₅Me₅)₂Sm(C₆H₅)(THF).