Small angle neutron scattering as sensitive tool to detect ligand-dependent shape changes in a plant lectin with β-trefoil folding and their dependence on the nature of the solvent

The galactoside-specific Viscum album L. agglutinin (VAA) is a potent biohazard akin to ricin and a mitogen for immune and tumor cells. These activities depend on cell surface binding to glycans. It is an open question whether the process of ligand binding alters the lectin's shape. Small angle neutron scattering (SANS) experiments revealed that the carbohydrate ligand lactose induced a decrease of the radius of gyration of dimeric VAA from 54.5 +/- 1 to 49.5 +/- 1 A in water. Apparently, VAA in aqueous solution and at the concentrations tested at 3.6 mg/ml and above adopts a compacted structure as response to ligand binding. In contrast to the behavior in aqueous solution, lactose binding in DMSO resulted in an increase of the lectin's radius of gyration from 49 +/- 1 to 55.5 +/- 1 A. Because shape changes may be reflected in the thermostability of the protein, this parameter was examined by activity assays of protein exposed to 60 degrees C and 70 degrees C and by differential scanning calorimetry (DSC). In line with the lactose-induced conformational alterations revealed by the SANS experiments, lactose presence enhanced the thermostability of VAA in water. Thus, binding of the carbohydrate ligand in solution can entail changes in shape and thermostability in the case of the tested plant lectin.     

Cryoenzymology of human plasmin catalysis: comparison of cryosolvents and reactions with nitrophenyl ester and anilide substrates

A comparative study of nucleophilic (methanol), aprotic (dimethyl sulfoxide), and protic but non nucleophilic (ethylene glycol, ethylene glycol/dimethylformamide) solvents on the catalytic and structural properties of human plasmin has been made. All four solvent systems are potentially suitable as cryosolvents for plasmin catalysis at subzero temperatures although the solubility of plasmin is limited in the methanol and dimethyl sulfoxide systems. Each cryosolvent system caused minor effects on the catalytic properties of the enzyme, which could be rationalized in terms of the known physical properties of the cosolvent. Solvent systems containing ethylene glycol induce a minor conformational change which increases the catalytic efficiency of plasmin. The cosolvent effects on Km and Ki indicate that electrostatic interactions dominate the binding of both substrates and inhibitors such as benzamidine. A change in slope of the Arrhenius plots for catalysis, reflecting a temperature-induced isomerization, is observed around 0 degree C; the energies of activation being 13 +/- 2 kcal mol-1 at higher temperatures and 19 +/- 2 kcal mol-1 at subzero temperatures, and essentially independent of solvent. Deacylation was shown to be the rate-limiting step in the hydrolysis of specific p-nitrophenyl ester substrates. Previous stopped-flow studies at room temperature provided observations suggesting that a tetrahedral intermediate could be detected in the plasmin-catalyzed hydrolysis of p-nitroanilide substrates. Experiments at subzero temperatures with such substrates failed to reveal any buildup of a tetrahedral intermediate under the experimental conditions.     

Evidence for major structural changes in the Manduca sexta midgut V1 ATPase due to redox modulation. A small angle X-ray scattering study

The shape and overall dimensions of the oxidized and reduced form of the V(1) ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6. 20 +/- 0.06 and 5.84 +/- 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 +/- 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V(1) molecule provide the first strong evidence for major structural changes in the V(1) ATPase because of redox modulation.     

An analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution.

The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. The structure of the purified receptor in detergent solution has previously been investigated by sedimentation analysis and gel filtration. Sedimentation analysis yielded a molecular weight of 250,000 for the protein moiety of the receptor monomer-detergent complex; hydrodynamic characteristics such as the Stokes radius, however, refer to the receptor-detergent complex. In this paper we report the results of our use of low-angle neutron scattering to investigate the shape of the receptor-detergent (Triton X-100 from Rohm & Haas Co., Philadelphia, Pa.) complex and separately of its protein and detergent moieties. By adjustment of the neutron-scattering density of the solvent with D2O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in approximately 18% D2O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +/- 1A. The radius of gyration and molecular volume (305,000 A3) of the receptor are inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) approximately 150 x approximately 50 A or an oblate cylinder, approximately 25 x approximately 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +/- 40,000), the extent of Triton X-100 binding in the complex (approximately 0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4.     

Solution structure and conformational changes of the Streptomyces chitin-binding protein (CHB1)

The shape and overall dimensions of the recently discovered Streptomyces alpha-chitin-binding protein, CHB1, were investigated by synchrotron radiation X-ray solution scattering. The radius of gyration and the maximum size of CHB1 were determined to be 1.75 +/- 0.03 nm and 6.0 +/- 0.2 nm, respectively. Using two independent ab initio approaches the low-resolution shape of the protein was found to consist of two domains, an elongated main globule with a length of about 4 nm and a foot-like domain of about 2 nm width. The structural and functional properties of CHB1 depend strongly on the presence of disulfide bonds; upon their reduction, the protein loses its affinity to chitin.     

Conformation of Lys-plasminogen and the kringle 1-3 fragment of plasminogen analyzed by small-angle neutron scattering

Native human Glu-plasminogen (Glu1-Asn791) was previously shown to have a radius of gyration of 39 A and a shape best described by a prolate ellipsoid [Mangel, W. F., Lin, B., & Ramakrishnan, V. (1990) Science 248, 69-73]. Upon occupation of a weak lysine-binding site, the shape reversibly changes to that best described by a Debye random coil with a radius of gyration of 56 A. Conversion from the closed to the open form is not accompanied by any change in secondary structure, hence the closed conformation is formed by interaction between domains, the five kringles and the protease domain, and this is abolished upon conversion to the open form. Here we analyzed by small-angle neutron scattering the conformations of human Lys-plasminogen (Lys78-Asn791) and the fragment K1-3 that contains the first three kringles of plasminogen (Tyr80-Val338 or Tyr80-Val354). The shape of Lys-plasminogen was best described by a Debye random coil with a radius of gyration of 51 A, and occupation of its lysine-binding sites by 6-aminohexanoic acid did not dramatically alter its conformation. Thus Lys-plasminogen was in the open form, similar to that of Glu-plasminogen with its lysine-binding sites occupied. The fragment K1-3 in the absence or presence of 6-aminohexanoic acid had a shape best described equally either by an elongated prolate ellipsoid or by a Debye random coil, with a radius of gyration of 29 A. Our model for the two forms of plasminogen is that, in the closed form, domain interaction generates a compact, almost globular, structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small-angle X-ray scattering on malate synthase from baker's yeast. The native substrate-free enzyme and enzyme-substrate complexes.

Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique. Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined. As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage. These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system. 1. For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm. Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder. The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm. Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape. 2. Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme. These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves). Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness. These changes imply an increase of the axial ratio by 2.2--6.9%, i.e. substrate binding induces a decrease of anisometry. While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter. In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA.     

Structural Insights into the A1 ATPase from the archaeon, Methanosarcina mazei Gö1

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei G?1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.     

Size of a human serum albumin molecule in solution]

The size of a human serum albumin molecule in aqueous solution containing 150 mM NaCl was studied using small-angle neutron scattering. The molecular radius of gyration was estimated to be 27.4 +/- 0.35 A. The compact sphere should have a smaller radius of gyration, whereas the popular human serum albumin model, a "cigar" 136 A long, should correspond to a greater radius of gyration. Possible shapes of the human serum albumin molecule which are in accordance with the results obtained, are the following: an extended ellipsoid less than 110 A of length or a nonsymmetrical oblate ellipsoid with a diameter of 85 A. The oblate ellipsoid might be close to the heart"-shaped structure of the crystalline human serum albumin molecule. The size of the albumin molecule does not change significantly as pH increases to 8.9. The possibility of the dynamic coexistence of various human serum albumin conformers in solution is discussed.     

Small-angle X-ray scattering study by synchrotron orbital radiation reveals that high molecular weight subunit of glutenin is a very anisotropic molecule.

Small-angle X-ray scattering of one high molecular weight (HMW) subunit of wheat glutenin was measured at protein concentration ranges from 1.0 to 10.0 mg/ml. The radius of gyration of whole particles, RO, in aq. 50% (v/v) 1-propanol and 0.1M acetic acid was 16.6 +/- 0.1nm and 22.8nm, respectively, and the corresponding radius of gyration of the cross-section, RC, was 2.82 +/- 0.02 nm and 2.23 +/- 0.01 nm, which indicate that the glutenin HMW subunit exists as very anisotropic particles in both solutions. The RO and RC values of the subunit, and the drastic decrease in scattered intensity at small angles that occurs in the acetic acid solution with relatively low protein concentration are completely explained in terms of rod-like molecules of the glutenin HMW subunit.     

Inhibition of the human erythrocyte calcium pump by dimethyl sulfoxide

The action of dimethyl sulfoxide on the human red cell Ca2+ pump was studied in inside-out vesicles. In a high-K+ medium at pH 7.6, the organic solvent inhibited both Ca2+ transport and ATP hydrolysis. Half-maximal effect was obtained with about 2% (v/v). At or below 10% dimethyl sulfoxide, the inhibition was overcome by adding inorganic phosphate or oxalate. In the absence of organic solvent, Ca2+ efflux from Ca(2+)-loaded vesicles consisted of a slow and a fast component whilst in its presence, there appears additionally a leakage component. The size of the latter depended markedly on dimethyl sulfoxide concentration, being about 3% at that level where Ca2+ uptake was half-maximally inhibited. ATP hydrolysis was more sensitive to dimethyl sulfoxide (10%) when free Ca2+ was increased within the millimolar level than when it was raised within the micromolar range. On the other hand, raising Ca2+ with organic solvent greatly stimulated ATP synthesis through ATP-Pi exchange, without reaching saturation. The results suggest that dimethyl sulfoxide blocks the red cell Ca2+ pump by increasing the affinity of the Ca2+ translocating site at the releasing step. They also show that at high concentrations, this solvent increases Ca2+ permeability.     

Inhibition of insulin receptor binding by dimethyl sulfoxide.

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 125-I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 125I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor was affected to a much lesser extent.     

Calcium-induced shape change of calmodulin with mastoparan studied by solution X-ray scattering

Solution x-ray scattering using synchrotron radiation as an x-ray source was used to analyze the Ca2+-dependent shape change of pig brain calmodulin in detail. The radius of gyration of calmodulin at 10 mg/ml was increased by 0.9 A. The increase was nearly completed when 2.5 mol of Ca2+/mol of calmodulin was added, whereas the radius of gyration of calmodulin with mastoparan decreased by about 3 A with an increasing Ca2+ concentration up to 4 mol of Ca2+/mol of calmodulin. At a moderate angle of region, both scattering profiles from calmodulin with or without Ca2+ displayed clear humps at s = 0.03 A-1 which are characteristic of a dumbbell structure. However, in the presence of mastoparan, the hump in the scattering profile became obscure and later disappeared with the third and fourth Ca2+ binding to calmodulin. These findings are attributable to the Ca2+-induced shape change of calmodulin with mastoparan from a dumbbell structure to a non-dumbbell structure in which the distance between the two lobes of calmodulin become closer by a bend in the central helix.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Bactericidal activity of peroxynitrite.

Peroxynitrite is a strong oxidant formed by macrophages and potentially by other cells that produce nitric oxide and superoxide. Peroxynitrite was highly bactericidal, killing Escherichia coli in direct proportion to its concentration with an LD50 of 250 microM at 37 degrees C in potassium phosphate, pH 7.4. The apparent bactericidal activity of a given concentration peroxynitrite at acidic pH was less than that at neutral and alkaline pH. However, after taking the rapid pH-dependent decomposition of peroxynitrite into account, the rate of the killing was not significantly different at pH 5 compared to pH 7.4. Metal chelators did not decrease peroxynitrite-mediated killing, indicating that exogenous transition metals were not required for toxicity. The hydroxyl radical scavengers mannitol, ethanol, and benzoate did not significantly affect toxicity while dimethyl sulfoxide enhanced peroxynitrite-mediated killing. Dimethyl sulfoxide is a more efficient hydroxyl radical scavenger than the other three scavengers and increased the formation of nitrogen dioxide from peroxynitrite. In the presence of 100 mM dimethyl sulfoxide, 60.0 +/- 0.3 microM nitrogen dioxide was formed from 250 microM peroxynitrite as compared to 2.0 +/- 0.1 microM in buffer alone. Thus, formation of nitrogen dioxide may have enhanced the toxicity of peroxynitrite decomposing in the presence of dimethyl sulfoxide.     

Inhibition of insulin receptor binding by dimethyl sulfoxide

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of ¹²⁵I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of ¹²⁵I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor were affected to a much lesser extent.     

A synchrotron X-ray scattering characterization of purified tubulin and of its expansion induced by mild detergent binding

This report presents a synchrotron radiation X-ray scattering characterization of calf brain tubulin purified by the modified Weisenberg procedure. The results show that under nonassembly conditions (i.e., in 10 mM sodium phosphate and 0.1 mM GTP, pH 7, buffer) these preparations consist of a uniform population of molecules with a radius of gyration of 3.1 +/- 0.1 nm, which can be interpreted as arising from the native alpha-beta heterodimer. The uniformity in the population persists even at unusually high concentrations of protein. Binding of colchicine or substitution of GTP by GDP does not induce, within the statistical accuracy and resolution range of our measurements, any significant structural modification in soluble tubulin. In assembly buffer [i.e., 10 mM sodium phosphate, 6 mM magnesium chloride, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 1 mM GTP, and 3.4 M glycerol, pH 6.5], these preparations readily assemble into microtubules upon increasing the temperature from 4 to 37 degrees C. Binding of nondenaturing amphiphiles to soluble tubulin provides a simplified model for tubulin-membrane interactions. The X-ray scattering data show that the radius of gyration of tubulin progressively increases upon binding of the mild detergent sodium deoxycholate, reaching a maximum value of 4.3 +/- 0.1 nm at detergent saturation. The relative increase in the radius of gyration coincides within experimental error with the previously determined relative increase in the frictional coefficient [Andreu, J.M., & Mu?oz, J.A. (1986) Biochemistry 25, 5220-5230]. Analysis of these observations suggests that the effect of detergent binding is to induce an isotropic swelling of the protein structure.     

Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle X-ray scattering

We have used solution small-angle X-ray scattering to characterize bovine brain calmodulin in the presence and absence of calcium. In the presence of calcium, calmodulin exists in solution as an elongated molecule with a radius of gyration of 21.5 A and a maximum vector length of approximately 62 A. These values are consistent with the dimensions recently determined for the crystal form of rat testis calmodulin. In the absence of calcium, the calmodulin molecule is shorter, the radius of gyration decreases to 20.6 A, and the maximum vector length decreases to approximately 58 A. This change in dimensions is consistent with an overall contraction of the protein through movement of the two lobes closer to each other upon removal of calcium from calmodulin.     

Yeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate.

Using small-angle X-ray scattering from solutions of yeast hexokinase, we have measured the radii of gyration of the monomeric B isozyme and its complexes with sugar substrates. We find that the radius of gyration decreases by 0.95 +/- 0.24 A upon binding glucose and 1.25 +/- 0.28 A upon binding glucose 6-phosphate. This observed reduction in radius of gyration in the presence of glucose is the same as that calculated from the coordinates of the high-resolution crystal structures of native hexokinase B and a glucose complex with hexokinase A. Thus, these measurements suggest that the dramatic closing of the slit between the two lobes of hexokinase observed in the crystal structures (Bennett, W.S., & Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848--4852) occurs in solution when either glucose or glucose 6-phosphate is bound.     

Membrane channel forming polypeptides. Molecular conformation and mitochondrial uncoupling activity of antiamoebin, an alpha-aminoisobutyric acid containing peptide

The conformations of the 16-residue fungal peptide antiamoebin I (Ac-Phe-Aib-Aib-Aib-D-Iva-Gly-Leu-Aib-Aib-Hyp-Gln-D-Iva-Hyp-Aib-Pro-P hol) have been investigated in dimethyl sulfoxide solution by one- and two-dimensional NMR techniques. A substantial number of resonances in the 270-MHz 1H NMR spectrum have been assigned. Intramolecularly hydrogen-bonded (solvent inaccessible) NH groups have been identified by determining solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. Ten backbone NH groups are inaccessible to solvent, while three NH groups assigned to the Phe(1), Aib(2), and Aib(8) residues are exposed to solvent. Interresidue nuclear Overhauser effects are consistent with psi values of approximately 120 +/- 30 degrees for Phe(1) and Leu(7). The NMR results, together with the stereochemical constraints imposed by the presence of alpha-aminoisobutyryl, isovalyl, prolyl, and 4-hydroxyprolyl residues, favor a highly ordered structure. Two backbone conformations consistent with the data are considered. Antiamoebin is shown to be an effective uncoupler of oxidative phosphorylation in rat liver mitochondria, providing evidence for its membrane-modifying activity.