Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods.

Water soluble polysaccharides from soybean (SSPS) have a pectin-like structure and are used as stabilisers in acidified beverages. Physicochemical properties such as structure, molecular weight and shape or conformation are primary factors controlling their functional properties. Two soybean polysaccharides, a native SSPS and a modified SSPS treated with beta-(1-->4)-D-galactosidase (GPase/SSPS) were studied by dynamic and static light scattering (DLS, SLS) and size exclusion chromatography (SEC). Consecutive filtrations using a range of membrane pore size removed a small fraction of macromolecular aggregates from dilute polysaccharide solutions with relatively little effect on the major component molecules as monitored by DLS and SEC measurements. Access to aggregate-free dilute solutions of SSPS and GPase/SSPS allowed the direct measurement of molecular characteristics. SLS results showed that SSPS had a weight average molecular weight of (645+/-11)x 10(3)g/mol and a radius of gyration, Rg, of (23.5+/-2.8)nm. By comparing R(g) with the hydrodynamic radius, Rh (21.1+/-0.5 nm) obtained from DLS, the structural parameter rho (Rg/Rh) was found to be 1.1, suggesting that SSPS has an overall globular shape due to a highly branched structure. The modified SSPS had a significantly lower molecular weight (287+/-18)x 10(3)g/mol but a similar radius of gyration (23.2+/-1.7 nm). The structure parameter rho of GPase/SSPS was higher (rho=1.3) because of a smaller hydrodynamic radius (17.7+/-1.8 nm). This suggests that GPase/SSPS has a much less branched structure yet still differs significantly from a linear random coil conformation (rho=1.7-2.0). The results derived from SLS and DLS are in agreement with the conclusions obtained from a chemical analysis where the reduction of molecular weight of GPase/SSPS was caused by the cleavage of galactan side chains.

Interaction of the 18.5-kD isoform of myelin basic protein with Ca2+ -calmodulin: effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism.

The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP-Cit(0)), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit(6)), human component C1 (hMBP-Cit(0)), and human component C8 (hMBP-Cit(6)), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit(0) and hMBP-Cit(0) bound CaM in a Ca(2+)-dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM-binding. Inherent Trp fluorescence spectroscopy and a single-site binding model were used to determine the dissociation constants: K(d) = 144 +/- 76 nM for rmMBP-Cit(0), and K(d) = 42 +/- 15 nM for hMBP-Cit(0). For rmMBP-qCit(6) and hMBP-Cit(6), the changes in fluorescence were suggestive of a two-site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady-state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.

Effects of the osmolyte trimethylamine-N-oxide on conformation, self-association, and two-dimensional crystallization of myelin basic protein.

The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring "chemical chaperone" that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys-->Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light-scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination.

Regioselective silylation of sugars through palladium nanoparticle-catalyzed silane alcoholysis.

Palladium(0)-catalyzed silane alcoholysis was applied to sugars for the first time using tert-butyldimethylsilane (TBDMS-H) and Ph(3)SiH as the silanes. The catalyst is a colloidal solution of Pd(0) generated in situ from PdX(2) (X = Cl(-), OAc(-)) and TBDMS-H in N,N-dimethylacetamide. The colloid has been characterized by dynamic light scattering and transmission electron microscopy and consists of catalytically highly active nanoparticles of approximately 2 nm diameter. The silane alcoholysis reaction is an effective method for the regioselective silylation of methyl and phenyl glycosides and generates hydrogen gas as the only side product. For many of the sugar substrates investigated, the distribution of regioisomers obtained is complementary to that of the traditional R(3)SiCl/base (base = pyridine, imidazole) methodology and gives convenient access to the 3,6- rather than the 2,6-silylated pyranosides, obtained as the main product by the silyl chloride method. The method also allows a selective axial silylation of levoglucosan and 1,3,5-O-methylidene-myo-inositol. In an attempt to rationalize the observed regioselectivities, ab initio predictions (HF/3-21G) have been made on the relative energies of some of the silylated products. They suggest that the observed regioselectivities do not reflect a kinetic vs thermodynamic product distribution but are induced by the silylation agent employed. Models for the possible origin of the observed regioselectivity in both silylation methods (silane- and silyl chloride-based) are discussed.

Osmotically induced shape changes of large unilamellar vesicles measured by dynamic light scattering.

Static and dynamic light scattering measurements have been used to characterize the size, size distribution, and shape of extruded vesicles under isotonic conditions. Dynamic light scattering was then used to characterize osmotically induced shape changes by monitoring changes in the hydrodynamic radius (R(h)) of large unilamellar vesicles (LUVs). These changes are compared to those predicted for several shapes that appear in trajectories through the phase diagram of the area difference elasticity (ADE) model (. Phys. Rev. E. 52:6623-6634). Measurements were performed on dioleoylphosphatidylcholine (DOPC) vesicles using two membrane-impermeant osmolytes (NaCl and sucrose) and a membrane-permeant osmolyte (urea). For all conditions, we were able to produce low-polydispersity, nearly spherical vesicles, which are essential for resolving well-defined volume changes and consequent shape changes. Hyper-osmotic dilutions of DOPC vesicles in urea produced no change in R(h), whereas similar dilutions in NaCl or sucrose caused reductions in vesicle volume resulting in observable changes to R(h). Under conditions similar to those of this study, the ADE model predicts an evolution from spherical to prolate then oblate shapes on increasing volume reduction of LUVs. However, we found that DOPC vesicles became oblate at all applied volume reductions.

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids.

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.

Small-angle neutron scattering from large unilamellar vesicles: an improved method for membrane thickness determination.

Small-angle neutron scattering (SANS) measurements were performed on large unilamellar vesicles (LUVs) in order to investigate solute effects on membrane properties. Although SANS is a well established technique for the measurement of membrane thickness in unilamellar vesicles, earlier measurements have depended on approximate treatments of the scattering function and have suffered from effects of multilamellarity or difficulty in sample preparation. More recent studies of temperature induced thickness changes in DPPC LUVs which have included explicit treatment of the full scattering function were complicated by disparities between the predicted and measured scattering curves. Here, we reexamine theoretical descriptions of SANS from LUVs. Motivated by our observations, we then introduce a new method for interpretation of SANS data, which we compare to established techniques and apply to our measurements.

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes.

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.

Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts.

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.

Small-angle light scattering: instrumental design and application to particle sizing.

A small-angle integrated light-scattering (SAILS) instrument was designed with the innovative use of a diffusing plate and a charge-coupled device (CCD) camera. In contrast to previous small-angle light-scattering instruments, SAILS has few optical surfaces, allowing the direct recovery of scattering data. Although this approach bypasses the need for aberration corrections that are due to lenses, geometric corrections still apply and are described in detail. The image on the diffusing plate, when photographed by the CCD camera, yields a digitized two-dimensional array, covering the observed scattering angles from 10 to 20 deg. The size distribution of the scattering particles can be obtained by a discrete inversion of the experimentally obtained intensity versus angle-scattering curve. The mean radii obtained from this inversion of SAILS data are compared with nominal sizes given by the manufacturer, and standard errors are computed. The results indicate that SAILS is an ideal instrument for the study of particulates and, because of its fast readout time, is suitable for studying aggregation phenomena. However, because of the limited Q range of SAILS it is currently not suited for the direct determination of particle diameters smaller than approximately 300 nm.

Optical changes in unilamellar vesicles experiencing osmotic stress.

Membrane properties that vary as a result of isotropic and transmembrane osmolality variations (osmotic stress) are of considerable relevance to mechanisms such as osmoregulation, in which a biological system "senses" and responds to changes in the osmotic environment. In this paper the light-scattering behavior of a model system consisting of large unilamellar vesicles of dioleoyl phosphatidyl glycerol (DOPG) is examined as a function of their osmotic environment. Osmotic downshifts lead to marked reductions in the scattered intensity, whereas osmotic upshifts lead to strong intensity increases. It is shown that these changes in the scattering intensity involve changes in the refractive index of the membrane bilayer that result from an alteration in the extent of hydration and/or the phospholipid packing density. By considering the energetics of osmotically stressed vesicles, and from explicit analysis of the Rayleigh-Gans-Debye scattering factors for spherical and ellipsoidal shells, we quantitatively demonstrate that although changes in vesicle volume and shape can arise in response to the imposition of osmotic stress, these factors alone cannot account for the observed changes in scattered intensity.

Spherical particle size determination by analytical inversion of the UV-visible-NIR extinction spectrum.

An analytic inversion method, based on the anomalous diffraction approximation for nonabsorbing spherical particles, was developed to retrieve the size distribution from the optical turbidity or extinction spectrum. This method makes use of a differential Fourier cosine transform approach and provides a simple and fast inversion by means of fast Fourier transform and the Savitzky-Golay filter. The applicability of this algorithm was tested on the extinction data generated by the Mie solution. The effects of noise, modality, band limits, and data set size were analyzed by comparison with simulated data. This method can be used to reconstruct the original monomodal and bimodal distributions from 10% noise-corrupted data. The peak position and ratio of peak heights can be recovered with 10% or less deviation. The experiments with latex spheres showed that the inversion result from this method compares favorably with that from the dynamic light scattering measurement.

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles.

Vesicle sizing by static light scattering: a Fourier cosine transform approach.

A Fourier cosine transform method, based on the Rayleigh-Gans-Debye thin-shell approximation, was developed to retrieve vesicle size distribution directly from the angular dependence of scattered light intensity. Its feasibility for real vesicles was partially tested on scattering data generated by the exact Mie solutions for isotropic vesicles. The noise tolerance of the method in recovering unimodal and biomodal distributions was studied with the simulated data. Applicability of this approach to vesicles with weak anisotropy was examined using Mie theory for anisotropic hollow spheres. Aprimitive theory about the first four moments of the radius distribution about the origin, excluding the mean radius, was obtained as an alternative to the direct retrieval of size distributions.

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.

Mechanical properties of vesicles. II. A model for osmotic swelling and lysis.

Vesicle polydispersity and leakage of solutes from the vesicle lumen influence the measurement and analysis of osmotically induced vesicle swelling and lysis, but their effects have not been considered in previous studies of these processes. In this study, a model is developed which expressly includes polydispersity and leakage effects. The companion paper demonstrated the preparation and characterization of large unilamellar lipid vesicles. A dye release technique was employed to indicate the leakage of solutes from the vesicles during osmotic swelling. Changes in vesicle size were monitored by dynamic light scattering (DLS). In explaining the results, the model identifies three stages. The first phase involves differential increases in membrane tension with strain increasing in larger vesicles before smaller ones. In the second phase, the yield point for lysis (leakage) is reached sequentially from large sizes to small sizes. In the final phase, the lumen contents and the external medium partially equilibrate under conditions of constant membrane tension. When fit to the data, the model yields information on polydispersity-corrected values for membrane area compressibility, Young's modulus, and yield point for lysis.

Determination of vesicle size distributions by freeze-fracture electron microscopy.

The most common electron microscopic technique for obtaining information on size distributions of uncollapsed membrane vesicles is based on the method of van Venetie (1980). This technique involves the sizing of only those vesicles that were freeze fractured at their equatorial planes. As a result, only a small number of images can be used to generate size distributions. Further, the technique is susceptible to systematic error. An alternate approach is to consider the complete distribution of image sizes and use this distribution to determine the average size and distribution of the vesicles. It is shown that the mean vesicle size is 4/pi times the mean image size. As well, a parameter, m, which can be determined from the image distribution, can be used to characterize the vesicle distribution. The advantage of this new approach is that images of all vesicles are used, leading to a statistically better determination of vesicle sizes.

Identification and characterization of nonsedimentable lipid-protein microvesicles.

Previously uncharacterized lipid-protein microvesicles have been isolated from young and senescing bean cotyledon tissue. The microvesicles are nonsedimentable and enriched in phospholipid degradation products (free fatty acids, long-chain aldehydes, and long-chain hydrocarbons). They range from 70 to 170 nm (radius) with a mean radius of 132 nm, and it is clear from freeze-fracture electron micrographs that they are bilayered in nature. Nonsedimentable lipid-protein microvesicles containing the same products of phospholipid degradation but smaller were also formed in vitro when smooth microsomal membranes from young cotyledon tissue were treated with Ca2+ to stimulate enzymatic degradation of phospholipids. The data suggest that these microvesicles comprise an intermediate stage of membrane lipid deterioration. They appear to serve as a vehicle for moving phospholipid degradation products out of membranes into the cytosol during senescence and perhaps also during normal membrane lipid turnover.

Vesicle sizing: Number distributions by dynamic light scattering.

A procedure is described which optimizes nonnegative least squares and exponential sampling fitting methods for analysis of dynamic light scattering (DLS) data from aqueous suspensions of vesicle/liposome systems. This approach utilizes a Rayleigh-Gans-Debye form factor for a coated sphere and yields number distributions which can be compared directly to distributions obtained by freeze-fracture electron microscopy (EM). Excellent agreement between the DLS and EM results are obtained for vesicle size distributions in the 100-200-nm range.

Dynamic instability of sheared microtubules observed by quasi-elastic light scattering.

The kinetics of microtubule reassembly was studied in vitro by quasi-elastic light scattering (QELS). When microtubules assembled in the absence of microtubule-associated proteins (MAPs) were sheared, they rapidly depolymerized, recovered, and reassembled. The mean length of the recovered microtubules was the same as that observed just before shearing, implying that on average one fragment per original microtubule survived the fragmentation and recovery. When microtubules that contained 25 percent brain MAP were sheared, the fragments did not depolymerize extensively and the average length of the fragments decreased by a factor of 3 relative to the unsheared sample. The results support the dynamic instability model, which predicts that cellular microtubules are latently unstable structures protected on their ends by stabilizing caps.