Small-angle neutron scattering study of the lipid bilayer thickness in unilamellar dioleoylphosphatidylcholine vesicles prepared by the cholate dilution method: n-decane effect

Previous X-ray diffraction studies on fully hydrated fluid lamellar egg phosphatidylcholine phases indicated a approximately 10 A increase of bilayer thickness in the presence of excess n-decane [Biochim. Biophys. Acta 597 (1980) 455], while the small-angle neutron scattering (SANS) on unilamellar extruded dioleoylphosphatidylcholine (DOPC) vesicles detected substantially smaller 2.4+/-1.3 A bilayer thickness increase at n-decane/DOPC molar ratio of 1.2 [Biophys. Chem. 88 (2000) 165]. The purpose of the present study is to investigate the n-decane effect on the bilayer thickness in unilamellar DOPC vesicles prepared by the sodium cholate (NaChol) dilution method. Mixed DOPC+NaChol micelles at DOPC and NaChol concentrations of 0.1 mol/l were prepared in 2H(2)O containing 0.135 mol/l NaCl. This micellar solution was diluted in 0.135 mol/l NaCl in 2H(2)O to reach the final DOPC and NaChol concentrations of 0.008 mol/l. Thirty microliters of n-decane solution in methanol was added to 1 ml of this dispersion. After methanol evaporation, SANS was conducted on the dispersions. From the Kratky-Porod plot ln[I(Q)Q(2)] vs. Q(2) of SANS intensity I(Q) in the range of scattering vector values Q corresponding to interval 0.001 A(-2)相似文献   

The kinetics of sodium extrusion in striated muscle as functions of the external sodium and potassium ion concentrations

After a 20 min initial washout, the rate of loss of radioactively labeled sodium ions from sodium-enriched muscle cells is sensitive to the external sodium and potassium ion concentrations. In the absence of external potassium ions, the presence of external sodium ions increases the sodium efflux. In the presence of external potassium ions, the presence of external sodium ions decreases the sodium efflux. In the absence of external potassium ions about one-third of the Na⁺ efflux that depends upon the external sodium ion concentration can be abolished by 10^-5 M glycoside. The glycoside-insensitive but external sodium-dependent Na⁺ efflux is uninfluenced by external potassium ions. In the absence of both external sodium and potassium ions the sodium efflux is relatively insensitive to the presence of 10^-5 M glycoside. The maximal external sodium-dependent sodium efflux in the absence of external potassium ions is about 20% of the magnitude of the maximal potassium-dependent sodium efflux. The magnitude of the glycoside-sensitive sodium efflux in K-free Ringer solution is less than 10% of that observed when sodium efflux is maximally activated by potassium ions. The inhibition of the potassium-activated sodium efflux by external sodium ions is of the competitive type. Reducing the external sodium ion concentration displaces the plots of sodium extrusion rate vs. [K]_o to the left and upwards.     

Crystal structures of aprotinin and its complex with sucrose octasulfate reveal multiple modes of interactions with implications for heparin binding

The crystal structures of aprotinin and its complex with sucrose octasulfate (SOS), a polysulfated heparin analog, were determined at 1.7-2.6 Å resolutions. Aprotinin is monomeric in solution, which associates into a decamer at high salt concentrations. Sulfate ions serve to neutralize the basic amino acid residues of aprotinin to stabilize the decameric aprotinin. Whereas SOS interacts with heparin binding proteins at 1:1 molar ratio, SOS was surprisingly found to induce strong agglutination of aprotinins. Five molecules of aprotinin interact with one molecule of the sulfated sugar, which is stabilized by electrostatic interactions between the positively charged residues of aprotinin and sulfate groups of SOS. The multiple binding modes of SOS with five individual aprotinin molecules may represent the diverse patterns of potential heparin binding to aprotinin, reflecting the interactions of densely packed protein molecules along the heparin polymer.     

Conformational flexibility of RecA protein filament: transitions between compressed and stretched states

RecA protein is a central enzyme in homologous DNA recombination, repair and other forms of DNA metabolism in bacteria. It functions as a flexible helix-shaped filament bound on stretched single-stranded or double-stranded DNA in the presence of ATP. In this work, we present an atomic level model for conformational transitions of the RecA filament. The model describes small movements of the RecA N-terminal domain due to coordinated rotation of main chain dihedral angles of two amino acid residues (Psi/Lys23 and Phi/Gly24), while maintaining unchanged the RecA intersubunit interface. The model is able to reproduce a wide range of observed helix pitches in transitions between compressed and stretched conformations of the RecA filament. Predictions of the model are in agreement with Small Angle Neutron Scattering (SANS) measurements of the filament helix pitch in RecA::ADP-AlF(4) complex at various salt concentrations.     

Effect of molecular weight distribution on the solution properties of sodium hyaluronate in 0.2M NaCl solution

Various molecular parameters, which characterize sodium hyaluronate in 0.2M NaCl solution, were obtained at 25°C by means of the static and dynamic light scattering and low shear viscometry over the molecular weight range of 5.94–627 × 10⁴. Molecular weight distribution was obtained by using the Laplace inversion method of the autocorrelation function of the scattered light intensity and by Yamakawa theory for the wormlike chain with the stiff chain parameters for sodium hyaluronate in 0.2M NaCl (persistence length, chain diameter, molar mass per unit contour length, and the excluded‐volume strength). The molecular weight distribution thus obtained reproduced the solution properties of sodium hyaluronate well. Especially, the intrinsic viscosity showed a good agreement over four orders of molecular weight with Yamakawa theory combined with the Barrett function. Sodium hyaluronate in 0.2M NaCl solution is well expressed by the wormlike chain model affected by the excluded‐volume effect with the persistence length of 4.2 nm. © 1999 John Wiley & Sons, Inc. Biopoly 50: 87–98, 1999     

The solution structure of full‐length dodecameric MCM by SANS and molecular modeling

The solution structure of the full‐length DNA helicase minichromosome maintenance protein from Methanothermobacter thermautotrophicus was determined by small‐angle neutron scattering (SANS) data together with all‐atom molecular modeling. The data were fit best with a dodecamer (dimer of hexamers). The 12 monomers were linked together by the B/C domains, and the adenosine triphosphatase (AAA+) catalytic regions were found to be freely movable in the full‐length dodecamer both in the presence and absence of Mg²⁺ and 50‐meric single‐stranded DNA (ssDNA). In particular, the SANS data and molecular modeling indicate that all 12 AAA+ domains in the dodecamer lie approximately the same distance from the axis of the molecule, but the positions of the helix–turn–helix region at the C‐terminus of each monomer differ. In addition, the A domain at the N‐terminus of each monomer is tucked up next to the AAA+ domain for all 12 monomers of the dodecamer. Finally, binding of ssDNA does not lock the AAA+ domains in any specific position, which leaves them with the flexibility to move both for helicase function and for binding along the ssDNA. Proteins 2014; 82:2364–2374. © 2014 Wiley Periodicals, Inc.     

A novel analytical ultracentrifugation based approach to the low resolution structure of gum arabic

Under investigation are the structural properties of gum arabic, an industrially important biopolymer for use as a stabilizer or in drug delivery, using Analytical Ultracentrifugation—a well‐established, matrix‐free probe for macromolecular size and shape. These results are combined with chromatographically‐coupled methods (multi‐angle light scattering, differential press imbalance viscometry) to provide a global analysis of its structure in varying ionic strength conditions. This analysis indicates that gum Arabic may have a compact, elliptical structure in solution, the significance of which for biotechnological use is indicated. This modelling method can be applied to other biopolymers and synthetic polymers. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 618–625, 2016.     

Conformational changes in single-strand DNA as a function of temperature by SANS

Small-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5'-ATGCTGATGC-3', in sodium phosphate buffer solution at 10 degrees C temperature increments from 25 degrees C to 80 degrees C. Cylindrical, helical, and random coil shape models were fitted to the SANS measurements at each temperature. All the shapes exhibited an expansion in the diameter direction causing a slightly shortened pitch from 25 degrees C to 43 degrees C, an expansion in the pitch direction with a slight decrease in the diameter from 43 degrees C to 53 degrees C, and finally a dramatic increase in the pitch and diameter from 53 degrees C to 80 degrees C. Differential scanning calorimeter scans of the sequence in solution exhibited a reversible two-state transition profile with a transition temperature of 47.5 +/- 0.5 degrees C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60 +/- 3 kJ mol(-1) that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47 +/- 1 degrees C was also obtained from ultraviolet optical density measurements of strand melting scans of the single-strand DNA. This transition corresponds to unstacking of the bases of the sequence and is responsible for the thermodynamic discrepancy between its binding stability to its complementary sequence determined directly at ambient temperatures and determined from extrapolated values of the melting of the duplex at high temperature.     

Effects of beta-cyclodextrin addition and temperature on the modulation of hydrophobic interactions in aqueous solutions of an associative alginate

Novel information about the effects of beta-cyclodextrin (beta-CD) addition and temperature on structural and rheological features of semidilute solutions of alginate and its hydrophobically modified analogue (HM-alginate) is given. Enhanced turbidity is observed for the HM-alginate solutions at high levels of beta-CD addition and low temperatures. The viscosity results revealed cross-linking of the alginate chains at high beta-CD concentrations and low temperatures. Rheological results for the HM-alginate solutions demonstrated that high levels of beta-CD addition and elevated temperatures promoted decoupling of the hydrophobic polymer-polymer associations via inclusion complex formation between beta-CD cavities and the hydrophobic side chains of the polymer. Analysis of small-angle neutron scattering (SANS) results from HM-alginate solutions in the presence of beta-CD suggested that the polymer chains are locally stretched at all of the considered levels of beta-CD and temperatures. The SANS results revealed association structures. The general picture that emerges is that beta-CD addition and temperature can be combined to tune the intensity of the hydrophobic interactions and to cross-link the unmodified alginate.     

Studying pressure denaturation of a protein by molecular dynamics simulations

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This “unfolding‐up‐on‐squeezing” is counter‐intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results—that pressure denatured states are water‐swollen, and theoretical results—that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states—their water‐swollen nature, retention of secondary structure, and overall compactness—mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure‐dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately ?60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500–2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water‐swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.     

Semi-Rigid Solution Structures of Heparin by Constrained X-ray Scattering Modelling: New Insight into Heparin-Protein Complexes

The anionic polysaccharides heparin and heparan sulphate play essential roles in the regulation of many physiological processes. Heparin is often used as an analogue for heparan sulphate. Despite knowledge of an NMR solution structure and 19 crystal structures of heparin-protein complexes for short heparin fragments, no structures for larger heparin fragments have been reported up to now. Here, we show that solution structures for six purified heparin fragments dp6-dp36 (where dp stands for degree of polymerisation) can be determined by a combination of analytical ultracentrifugation, synchrotron X-ray scattering, and constrained modelling. Analytical ultracentrifugation velocity data for dp6-dp36 showed sedimentation coefficients that increased linearly from 1.09 S to 1.84 S with size. X-ray scattering of dp6-dp36 gave radii of gyration R_G that ranged from 1.33 nm to 3.12 nm and maximum lengths that ranged from 3.0 nm to 12.3 nm. The higher resolution of X-ray scattering revealed an increased bending of heparin with increased size. Constrained molecular modelling of 5000 randomised heparin conformers resulted in 9-15 best-fit structures for each of dp18, dp24, dp30, and dp36 that indicated flexibility and the presence of short linear segments in mildly bent structures. Comparisons of these solution structures with crystal structures of heparin-protein complexes revealed similar ranges of phi (φ) and psi (ψ) angles between iduronate and glucosamine rings. We conclude that heparin in solution has a semi-rigid and extended conformation that is preformed for its optimal binding to protein targets without major conformational changes.     

Effects of bilirubin on potassium (86Rb+) influx and ionic content in Ehrlich ascites cells

Potassium influx, intracellular potassium and sodium content and cellular volume were determined in vitro in Ehrlich ascites cells in the presence of up to 0.8 mM bilirubin in the incubation medium. Bilirubin uptake into cells as a function of bilirubin concentration in the incubation medium increased linearly with a molar bilirubin/albumin ratio of 20 : 1. Potassium influx and intracellular content decreased while cellular volume increased after 180 min of incubation of cells in bilirubin at a molar bilirubin/albumin ratio of 20 : 1. At a bilirubin/albumin ratio 2 : 1, potassium influx decreased, cellular volume remained unchanged, and bilirubin uptake into cells became saturated at bilirubin concentrations greater than 0.3 mM. It is suggested that bilirubin-induced alterations in potassium gradients across cell membranes may play a role in toxic effects of bilirubin on cells.     

Calcium oscillations induced by gambierol in cerebellar granule cells

Gambierol is a marine polyether ladder toxin derived from the dinoflagellate Gambierdiscus toxicus. To date, gambierol has been reported to act either as a partial agonist or as an antagonist of sodium channels or as a blocker of voltage‐dependent potassium channels. In this work, we examined the cellular effect of gambierol on cytosolic calcium concentration, membrane potential and sodium and potassium membrane currents in primary cultures of cerebellar granule cells. We found that at concentrations ranging from 0.1 to 30 µM, gambierol‐evoked [Ca²⁺]c oscillations that were dependent on the presence of extracellular calcium, irreversible and highly synchronous. Gambierol‐evoked [Ca²⁺]c oscillations were completely eliminated by the NMDA receptor antagonist APV and by riluzole and delayed by CNQX. In addition, the K⁺ channel blocker 4‐aminopyridine (4‐AP)‐evoked cytosolic calcium oscillations in this neuronal system that were blocked by APV and delayed in the presence of CNQX. Electrophysiological recordings indicated that gambierol caused membrane potential oscillations, decreased inward sodium current amplitude and decreased also outward IA and IK current amplitude. The results presented here point to a common mechanism of action for gambierol and 4‐AP and indicate that gambierol‐induced oscillations in cerebellar neurons are most likely secondary to a blocking action of the toxin on voltage‐dependent potassium channels and hyperpolarization of sodium current activation. J. Cell. Biochem. 110: 497–508, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of bilirubin on potassium (86Rb+) influx and ionic content in Ehrlich ascites cells.

Potassium influx, intracellular potassium and sodium content and cellular volume were determined in vitro in Ehrlich ascites cells in the presence of up to 0.8 mM bilirubin in the incubation medium. Bilirubin uptake into cells as a function of bilirubin concentration in the incubation medium increased linearly with a molar bilirubin/albumin ratio of 20 : 1. Potassium influx and intracellular content decreased while cellular volume increased after 180 min of incubation of cells in bilirubin at a molar bilirubin/albumin ratio of 20 : 1. At a bilirubin/albumin ratio 2 : 1, potassium influx decreased, cellular volume remained unchanged, and bilirubin uptake into cells became saturated at bilirubin concentrations greater than 0.3 mM. It is suggested that bilirubin-induced alterations in potassium gradients across cell membranes may play a role in toxic effects of bilirubin on cells.     

Development and effectiveness of a soluble manganese silicate compound in controlling manganese deficiency in plants

Summary A study was made of soluble manganese silicates prepared by adding a solution of manganese sulfate to different concentrations of sodium silicate in alkaline medium. The solubility of manganese increased with increasing amounts of sodium silicate and reached its maximum at a molar Na₂SiO₃/MnSO₄ ratio between 10 and 15. At molar Na₂SiO₃/MnSO₄ ratios of 5 and more a red-brown coloured manganese silicate is formed, which is stable in the pH range of 4 to 12. Gel chromatographic analysis indicated that this compound contains 1 molecule Mn per 15 molecules SiO₂. At a molar Na₂SiO₃/MnSO₄ ratio of 15 the yield of soluble manganese could be raised to 98.5% of the theoretical amount by preventing the rapid oxidation of the Mn²⁺-ions in the alkaline solution. A pot experiment with oats was conducted to evaluate the effectiveness of the red-brown manganese silicate in controlling manganese deficiency. The effect of the Mn-carrier on Mn-content and yield of grain and on the colour scores was compared with that of Mn-EDTA, Mn-DTPA and MnSO₄·4H₂O. The manganese silicate was found to be a more effective manganese source than Mn-EDTA, Mn-DTPA or MnSO₄·4H₂O.     

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.     

The N-terminal module of HPV16 E7 is an intrinsically disordered domain that confers conformational and recognition plasticity to the oncoprotein

The HPV16 E7 oncoprotein is an extended dimer, with a stable and cooperative fold, but that displays properties of "natively unfolded" proteins. Two regions of conserved sequence are found in E7 proteins, where the N-terminus (1-40) includes the retinoblastoma tumor suppressor binding and casein kinase II phosphorylation sites. A fragment containing the highly acidic N-terminal half shows an apparently disordered conformation by far-UV-circular dichroism (CD) at neutral pH, and its hydrodynamic radius is much larger than a neutral peptide of the same length. Trifluoroethanol and micellar concentrations of sodium dodecyl sulfate stabilize a much more helical structure at pH 4.0 than at pH 7.5, while submicellar concentrations of the detergent yield a beta-strand. The shape, pH, and temperature dependence of the CD spectrum at pH 7.5 are indicative of a poly proline type II structure. This structure is stabilized by phosphorylation, which would translate into increased transforming activity in the cell. Thus, the intrinsically disordered properties of the N-terminal module of E7 are responsible for the structural plasticity of the oncoprotein. Although the domain is not a compact and cooperatively folded unit, it is a bona fide functional domain, evolved to maintain a dynamic but extended structure in the cell. These properties allow adaptation to a variety of protein targets and expose the PEST degradation sequence that regulates its turnover in the cell, a modification of which leads to the accumulation of E7 species with consequences in the transformation process.     

Small angle neutron and X-ray scattering in structural biology: recent examples from the literature

Small angle scattering can provide unique structural information on the shape, domain organisation, and interactions of biomacromolecules in solution. Small angle neutron scattering (SANS) combined with deuterium labelling makes it possible to define the positions of specific components within a complex while small angle X-ray scattering (SAXS) provides more precise data on the overall shape. Here I review four recent publications, three of which were presented at the Neutrons in Biology meeting at the STFC Rutherford Appleton Laboratory in July 2007, that utilise SANS, SAXS, and complementary techniques to define the solution structure of large multidomain proteins and macromolecular complexes. These four papers emphasise the critical importance of sample quality and characterisation as well as the important role played by complementary techniques in building structural models based on small angle scattering data. They show the ability of SANS and SAXS in determining solution structures provides an important complementary structural technique for large, flexible, and glycosylated proteins where high resolution structural techniques, such as crystallography and NMR, cannot be applied.