Supramolecular structures of peptide assemblies in membranes by neutron off-plane scattering: method of analysis. (1/232)

In a previous paper (Yang et al., Biophys. J. 75:641-645, 1998), we showed a simple, efficient method of recording the diffraction patterns of supramolecular peptide assemblies in membranes where the samples were prepared in the form of oriented multilayers. Here we develop a method of analysis based on the diffraction theory of two-dimensional liquids. Gramicidin was used as a prototype model because its pore structure in membrane in known. At full hydration, the diffraction patterns of alamethicin and magainin are similar to gramicidin except in the scale of q (the momentum transfer of scattering), clearly indicating that both alamethicin and magainin form pores in membranes but of different sizes. When the hydration of the multilayer samples was decreased while the bilayers were still fluid, the in-plane positions of the membrane pores became correlated from one bilayer to the next. We believe that this is a new manifestation of the hydration force. The effect is most prominent in magainin patterns, which are used to demonstrate the method of analysis. When magainin samples were further dehydrated or cooled, the liquid-like diffraction turned into crystal-like patterns. This discovery points to the possibility of investigating the supramolecular structures with high-order diffraction.  (+info)

Squalane is in the midplane of the lipid bilayer: implications for its function as a proton permeability barrier. (2/232)

A recently proposed model for proton leakage across biological membranes [Prog. Lipid Res. 40 (2001) 299] suggested that hydrocarbons specifically in the center of the lipid bilayer inhibit proton leaks. Since cellular membranes maintain a proton electrochemical gradient as a principal energy transducer, proton leakage unproductively consumes cellular energy. Hydrocarbons in the bilayer are widespread in membranes that sustain such gradients. The alkaliphiles are unique in that they contain up to 40 mol% isoprenes in their membranes including 10-11 mol% squalene [J. Bacteriol. 168 (1986) 334]. Squalene is a polyisoprene hydrocarbon without polar groups. Localizing hydrocarbons in lipid bilayers has not been trivial. A myriad of physical methods including fluorescence spectroscopy, electron-spin resonance, nuclear magnetic resonance as well as X-ray and neutron diffraction have been used to explore this question with various degrees of success and often contradictory results. Seeking unambiguous evidence for the localization of squalene in membranes or lipid bilayers, we employed neutron diffraction. We incorporated 10 mol% perdeuterated or protonated squalane, an isosteric analogue of squalene, into stacked bilayers of dioleoyl phosphatidyl choline (DOPC) doped with dioleoyl phosphatidyl glycerol (DOPG) to simulate the negative charges found on natural membranes. The neutron diffraction data clearly show that the squalane lies predominantly in the bilayer center, parallel to the plane of the membrane.  (+info)

Neutron and X-ray scattering by ox corneal stroma differentially loaded with bound anions. (3/232)

Ox corneas at near physiological hydration were subjected to two variables: the amount of chloride ions bound to them and exposure of various mixtures of H(2)O/D(2)O as solvent. The preparations were then exposed to a neutron beam and the contrast match points, at which the collagen fibrils of the corneal stroma most nearly matched the scattering density of the various H(2)O/D(2)O mixtures, were measured. In both cases of high and low bound chloride, the contrast match points of the collagen fibril were equal, indicating that there were no significant changes in the water of electrostriction at the fibril surface when chloride ions bind to the stroma. The data suggest that the ligands which bind anions to corneal stroma are not located at the collagen fibril surface. When the chloride binding ligands were extracted from the corneal stroma there were significant changes in the structure of the fibrils. We suggest that the chloride binding ligands may be located within the collagen fibril.  (+info)

Small-angle neutron scattering study of the lipid bilayer thickness in unilamellar dioleoylphosphatidylcholine vesicles prepared by the cholate dilution method: n-decane effect. (4/232)

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)+info)

Refolding of a high molecular weight protein: salt effect on collapse. (5/232)

Small-angle neutron scattering experiments were performed on dilute solutions of a high molecular weight protein (fibronectin, M = 580 kg/mol) in four cases: native conditions; unfolded state obtained by a denaturing agent (urea); and two badly refolded (or collapsed) states obtained by progressive elimination of the denaturing agent in salt-containing or salt-free solutions. Our main result is concerned by the conformation of the protein as the attempt for refolding is driven with or without salt. In salt-containing solution, we observe unambiguously that the protein chain collapses at large length scales but still obeys to a Gaussian statistics at short length scales. In other words, the globule embodies a large quantity of solvent compared to the compact situation. In salt-free solutions, the badly refolded protein is not globular but displays both a coil-like and an open conformation at large length scales and a local high density area. This behavior is discussed with respect to the scaling theories for polymers and polyampholytes.  (+info)

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy. (6/232)

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.  (+info)

Detection of ligand- and solvent-induced shape alterations of cell-growth-regulatory human lectin galectin-1 in solution by small angle neutron and x-ray scattering. (7/232)

The bioactivity of galectin-1 in cell growth regulation and adhesion prompted us to answer the questions whether ligand presence and a shift to an aprotic solvent typical for bioaffinity chromatography might alter the shape of the homodimeric human lectin in solution. We used small angle neutron and synchrotron x-ray scattering studies for this purpose. Upon ligand accommodation, the radius of gyration of human galectin-1 decreased from 19.1 +/- 0.1 A in the absence of ligand to 18.2 +/- 0.1 A. In the aprotic solvent dimethyl sulfoxide, which did not impair binding capacity, galectin-1 formed dimers of a dimer, yielding tetramers with a cylindrical shape. Intriguingly, no dissociation into subunits occurred. In parallel, NMR monitoring was performed. The spectral resolution was in accord with these data. In contrast to the properties of the human protein, a nonhomologous agglutinin from mistletoe sharing galactose specificity was subject to a reduction in the radius of gyration from approximately 62 A in water to 48.7 A in dimethyl sulfoxide. Evidently, the solvent caused opposite responses in the two tested galactoside-binding lectins with different folding patterns. We have hereby proven that ligand presence and an aprotic solvent significantly affect the shape of galectin-1 in solution.  (+info)

The membrane bound N-terminal domain of human adenosine diphosphate ribosylation factor-1 (ARF1). (8/232)

The small G protein adenosine diphosphate ribosylation factor-1 (ARF1) is activated by cell membrane binding of a self-folding N-terminal domain. We present a model of the human ARF1 N-terminal peptide in planar lipid bilayers, determined from neutron lamellar diffraction and circular dichroism data with molecular modelling. This amphipathic domain lies at a shallow membrane depth, ideal for regulation of the ARF1 bio-timer by rapid, reversible membrane binding. The helical region does not elongate upon membrane binding, leaving the connecting flexible linker region's length unchanged.  (+info)