In JoVE (1)

Other Publications (17)

Articles by Marie-Sousai Appavou in JoVE

 JoVE Bioengineering

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

1Jülich Centre for Neutron Science Outstation at MLZ, Forschungszentrum Jülich GmbH, 2Department of Chemistry, Louisiana State University, 3Jülich Centre for Neutron Science JCNS-1 & Institute of Complex Systems ICS-1, Forschungszentrum Jülich GmbH, 4Central Institute of Engineering, Electronics and Analytics — Electronic Systems (ZEA-2), Forschungszentrum Jülich GmbH, 5Central Institute of Engineering, Electronics and Analytics — Engineering and Technology (ZEA-1), Forschungszentrum Jülich GmbH


JoVE 54639

Other articles by Marie-Sousai Appavou on PubMed

Using Polarization Analysis to Separate the Coherent and Incoherent Scattering from Protein Samples

Biochimica Et Biophysica Acta. Jan, 2010  |  Pubmed ID: 19595800

Polarization analysis was used to separate experimentally the coherent and spin-incoherent nuclear static scattering functions, from a representative set of samples of interest for protein studies. This method had so far limited application in the study of amorphous materials, despite the relevance of the information that it provides. It allows, for instance, the experimental determination of the structure factor of materials containing a significant amount of hydrogen atoms, avoiding the contamination of measurements by a non-negligible incoherent background. Knowledge of the relative importance of the coherent and incoherent terms at different Q-values is also a pre-requisite for the interpretation of quasielastic neutron scattering experiments, performed at instruments in which the total dynamic scattering function is measured, such as conventional time-of-flight and backscattering spectrometers. Combining data from different instruments, it was possible to cover a wide Q-range, from the small-angle region (0.006

The Influence of 2 Kbar Pressure on the Global and Internal Dynamics of Human Hemoglobin Observed by Quasielastic Neutron Scattering

European Biophysics Journal : EBJ. Jun, 2011  |  Pubmed ID: 21340585

Pressure is a ubiquitous physical parameter in life and is commonly used in the life sciences to study new protein folding pathways or association-dissociation phenomena. In this paper, an investigation of the influence of pressure on hemoglobin, a multimeric protein, at the picosecond time scale is presented using time-of-flight neutron scattering. The aim is to observe the influence of pressure on the translational diffusion and internal motions of hemoglobin in a concentrated solution and a possible dissociation of the subunits as suggested by Pin et al. (Biochemistry 29:9194, 1990) using fluorescence spectroscopy. A new flat 2 kbar pressure cell made of an aluminum alloy has been used, which allowed the effect of pressure to be studied with minimum background contribution. Within this range of pressure, the effect of this physical parameter on global diffusion can be explained in terms of the change in the water buffer viscosity and an oligomerization of hemoglobin subunits, whereas the internal motions were less affected.

Colloidal Structure and Stability of DNA/polycations Polyplexes Investigated by Small Angle Scattering

Biomacromolecules. Dec, 2011  |  Pubmed ID: 22074384

Polyplexes of short DNA-fragments (300 b.p., 100 nm) with tailor-made amine-based polycations of different architectures (linear and hyperbranched) were investigated in buffer solution as a function of the mixing ratio with DNA. The resulting dispersed polyplexes were characterized using small-angle neutron and X-ray scattering (SANS, SAXS) as well as cryo-TEM with respect to their mesoscopic structure and their colloidal stability. The linear polyimines form rather compact structures that have a high tendency for precipitation. In contrast, the hyperbranched polycation with enzymatic-labile pentaethylenehexamine arms (PEHA) yields polyplexes colloidally stable for months. Here the polycation coating of DNA results in a homogeneous dispersion based on a fractal network with low structural organization at low polycation amount. With increasing polycation, bundles of tens of aligned DNA rods appear that are interconnected in a fractal network with a typical correlation distance on the order of 100 nm, the average length of the DNA used. With higher organization comes a decrease in stability. The 3D network built by these beams can still exhibit some stability as long as the material concentration is large enough, but the structure collapses upon dilution. SAXS shows that the complexation does not affect the local DNA structure. Interestingly, the structural findings on the DNA polyplexes apparently correlate with the transfection efficiency of corresponding siRNA complexes. In general, these finding not only show systematic trends for the colloid stability, but may allow for rational approaches to design effective transfection carriers.

Temperature-regulated Fluorescence and Association of an Oligo(ethyleneglycol)methacrylate-based Copolymer with a Conjugated Polyelectrolyte--the Effect of Solution Ionic Strength

The Journal of Physical Chemistry. B. Nov, 2013  |  Pubmed ID: 24143872

Aqueous mixtures of a dye-labeled non-ionic thermoresponsive copolymer and a conjugated cationic polyelectrolyte are shown to exhibit characteristic changes in fluorescence properties in response to temperature and to the presence of salts, enabling a double-stimuli responsiveness. In such mixtures at room temperature, i.e., well below the lower critical solution temperature (LCST), the emission of the dye is strongly quenched due to energy transfer to the polycation, pointing to supramolecular interactions between the two macromolecules. Increasing the concentration of salts weakens the interpolymer interactions, the extent of which is simultaneously monitored from the change in the relative emission intensity of the components. When the mixture is heated above its LCST, the transfer efficiency is significantly reduced, signaling a structural reorganization process, however, surprisingly only if the mixture contains salt ions. To elucidate the reasons behind such thermo- and ion-sensitive fluorescence characteristics, we investigate the effect of salts of alkali chlorides, in particular of NaCl, on the association behavior of these macromolecules before and after the polymer phase transition by a combination of UV-vis, fluorescence, and (1)H NMR spectroscopy with light scattering and small-angle neutron scattering measurements.

Softening of Phospholipid Membranes by the Adhesion of Silica Nanoparticles--as Seen by Neutron Spin-echo (NSE)

Nanoscale. Jun, 2014  |  Pubmed ID: 24838980

The interactions between nanoparticles and vesicles are of significant interest both from a fundamental as well as from a practical point of view, as vesicles can serve as a model system for cell membranes. Accordingly the effect of nanoparticles that bind to the vesicle bilayer is very important with respect to understanding their biological impact and also may shed some light on the mechanisms behind the effect of nanotoxicity. In this study we have investigated the influence of small adsorbed silica nanoparticles (SiNPs) on the structure of zwitterionic DOPC vesicles. By a combination of SANS, cryo-TEM, and DLS, we observed that the SiNPs are bound to the outer vesicle surface without significantly affecting the vesicle structure. Most interestingly, by means of neutron spin-echo (NSE) local bilayer fluctuations were studied and one finds a small but marked decrease of the membrane rigidity upon binding of the nanoparticles. This surprising finding may be a relevant aspect for the further understanding of the effects that nanoparticles have on phospholipid bilayers.

Oil-in-water Microemulsion Droplets of TDMAO/decane Interconnected by the Telechelic C18-EO150-C18: Clustering and Network Formation

Soft Matter. Jul, 2014  |  Pubmed ID: 24901947

The effect of a doubly hydrophobically end-capped water soluble polymer (C18-PEO150-C18) on the properties of an oil-in-water (O/W) droplet microemulsion (R ∼ 2.85 nm) has been studied as a function of the amount of added telechelic polymer. Macroscopically one observes a substantial increase of viscosity once a concentration of ∼5 hydrophobic stickers per droplet is surpassed and effective cross-linking of the droplets takes place. SANS measurements show that the size of the individual droplets is not affected by the polymer addition but it induces attractive interactions at low concentration and repulsive ones at high polymer content. Measurements of the diffusion coefficient by DLS and FCS show increasing sizes at low polymer addition that can be attributed to the formation of clusters of microemulsion droplets interconnected by the polymer. At higher polymer content the network formation leads to an additional slow relaxation mode in DLS that can be related to the rheological behaviour, while the self-diffusion observed in FCS attains a lower plateau value, i.e., the microemulsion droplets remain effectively fixed within the network. The combination of SANS, DLS, and FCS allows us to derive a self-consistent picture of the evolution of structure and dynamics of the mixed system microemulsion/telechelic polymer as a function of the polymer content, which is not only relevant for controlling the macroscopic rheological properties but also with respect to the internal dynamics as it is, for instance, relevant for the release and transport of active agents.

Surfactant or Block Copolymer Micelles? Structural Properties of a Series of Well-defined N-alkyl-PEO Micelles in Water Studied by SANS

Soft Matter. Jul, 2014  |  Pubmed ID: 24916456

Here we present an extensive small-angle neutron scattering (SANS) structural characterization of micelles formed by poly(ethylene oxide)-mono-n-alkyl ethers (Cn-PEOx) in dilute aqueous solution. Chemically, Cn-PEOx can be considered as a hybrid between a low-molecular weight surfactant and an amphiphilic block copolymer. The present system, prepared through anionic polymerization techniques, is better defined than other commercially available polymers and allows a very precise and systematic testing of the theoretical predictions from thermodynamical models. The equilibrium micellar properties were elaborated by systematically varying the n-alkyl chain length (n) at constant PEO molecular weight or increasing the soluble block size (x), respectively. The structure was reminiscent of typical spherical star-like micelles i.e. a constant core density profile, ∼r(0), and a diffuse corona density profile, ∼r(-4/3). Through a careful quantitative analysis of the scattering data, it is found that the aggregation number, Nagg initially rapidly decreases with increasing PEO length until it becomes independent at higher PEO molecular weight as expected for star-like micelles. On the other hand, the dependency on the n-alkyl length is significantly stronger than that expected from the theories for star-like block copolymer micelles, Nagg ∼ n(2) similar to what is expected for surfactant micelles. Hence the observed aggregation behavior suggests that the Cn-PEOx micelles exhibit a behavior that can be considered as a hybrid between low-molecular weight surfactant micelles and diblock copolymer micelles.

Structure and Dynamics of a Compact State of a Multidomain Protein, the Mercuric Ion Reductase

Biophysical Journal. Jul, 2014  |  Pubmed ID: 25028881

The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.

Noncanonical Self-assembly of Highly Asymmetric Genetically Encoded Polypeptide Amphiphiles into Cylindrical Micelles

Nano Letters. Nov, 2014  |  Pubmed ID: 25268037

Elastin-like polypeptides (ELPs) are a class of biopolymers consisting of the pentameric repeat (VPGαG)n based on the sequence of mammalian tropoelastin that display a thermally induced soluble-to-insoluble phase transition in aqueous solution. We have discovered a remarkably simple approach to driving the spontaneous self-assembly of high molecular weight ELPs into nanostructures by genetically fusing a short 1.5 kDa (XGy)z assembly domain to one end of the ELP. Classical theories of self-assembly based on the geometric mass balance of hydrophilic and hydrophobic block copolymers suggest that these highly asymmetric polypeptides should form spherical micelles. Surprisingly, when sufficiently hydrophobic amino acids (X) are presented in a periodic sequence such as (FGG)8 or (YG)8, these highly asymmetric polypeptides self-assemble into cylindrical micelles whose length can be tuned by the sequence of the morphogenic tag. These nanostructures were characterized by light scattering, tunable resistive pulse sensing, fluorescence spectrophotometry, and thermal turbidimetry, as well as by cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS). These short assembly domains provide a facile strategy to control the size, shape, and stability of stimuli responsive polypeptide nanostructures.

Long Wavelength Undulations Dominate Dynamics in Large Surfactant Membrane Patches

Nanoscale. Feb, 2015  |  Pubmed ID: 25579866

By exposing microemulsions to small (80 nm diameter) and large (500 nm) disk shaped clay particles we were able to show the presence of long wavelength undulations that only occur for large membrane patches. A combination of small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments have been applied to study microemulsions. These, consisting of D2O, d-decane and the surfactant C10E4, were used in connection with Laponite (small) and Nanofil (large) clay. To our knowledge our experiments show for the first time that the clay platelets induce lamellar ordering adjacent to the clay discs in the otherwise bicontinuous microemulsion. This is due to the fact that in purely structural investigations, radial averaging smears out the signature of the lamellar phase. For thermodynamically fluctuating membranes near interfaces the theory of Seifert predicts a cross-over of the dispersion relationship from k(2) to a k(3)-dependence. With the correlation length of the membrane patches being confined by the dimension of the clay platelets we were able to show that this in fact takes place but is only present for the larger Nanofil particles.

Phase Behavior of Nonionic Microemulsions with Multi-end-capped Polymers and Its Relation to the Mesoscopic Structure

Langmuir : the ACS Journal of Surfaces and Colloids. May, 2015  |  Pubmed ID: 25879684

The polymer architecture of telechelic or associative polymers has a large impact on the bridging of self-assembled structures. This work presents the phase behavior, small angle neutron scattering (SANS), dynamic light scattering (DLS), and fluorescence correlation spectroscopy (FCS) of a nonionic oil-in-water (O/W) microemulsion with hydrophobically end-capped multiarm polymers with functionalities f = 2, 3, and 4. For high polymer concentrations and large average interdroplet distance relative to the end-to-end distance of the polymer, d/R(ee), the system phase separates into a dense, highly connected droplet network phase, in equilibrium with a dilute phase. The extent of the two-phase region is larger for polymers with similar length but higher f. The interaction potential between the droplets in the presence of polymer has both a repulsive and an attractive contribution as a result of the counterbalancing effects of the exclusion by polymer chains and bridging between droplets. This study experimentally demonstrates that higher polymer functionalities induce a stronger attractive force between droplets, which is responsible for a more extended phase separation region, and correlate with lower collective droplet diffusivities and higher amplitude of the second relaxation time in DLS. The viscosity and the droplet self-diffusion obtained from FCS, however, are dominated by the end-capped chain concentration.

Simultaneous Phase Transfer and Surface Modification of TiO₂ Nanoparticles Using Alkylphosphonic Acids: Optimization and Structure of the Organosols

Langmuir : the ACS Journal of Surfaces and Colloids. Oct, 2015  |  Pubmed ID: 26421961

An original protocol of simultaneous surface modification and transfer from aqueous to organic phases of anatase TiO2 nanoparticles (NPs) using alkylphosphonic acids (PAs) is studied. The influence of the solvent, the nature and concentration of the PA, and the size, concentration, and aggregation state of the TiO2 NPs was investigated. Complete transfer was observed for linear alkyl chains (5, 8, 12, and 18 C atoms), even at very high sol concentrations. After transfer, the grafted NPs were characterized by (31)P solid-state MAS NMR. The dispersion state of NPs before and after phase transfer was monitored by dynamic light scattering (DLS). Small-angle neutron scattering (SANS) was used to characterize the structure of PA-grafted NPs in the organic solvent. Using a quantitative core-shell model cross-checked under different contrast conditions, it is found that the primary particles making up the NPs are homogeneously grafted with a solvated PA-layer. The nanometric thickness of the latter is shown to increase with the length of the linear carbon chain of the PA, independent of the size of the primary TiO2 NP. Interestingly, a reversible temperature-dependent aggregation was evidenced visually for C18PA, and confirmed by DLS and SANS: heating the sample induces the breakup of aggregates, which reassemble upon cooling. Finally, in the case of NPs agglomerated by playing with the pH or the salt concentration of the sols, the phase transfer with PA is capable of redispersing the agglomerates. This new and highly versatile method of NP surface modification with PAs and simultaneous transfer is thus well suited for obtaining well-dispersed grafted NPs.

Water Dynamics in Shewanella Oneidensis at Ambient and High Pressure Using Quasi-Elastic Neutron Scattering

Scientific Reports. Jan, 2016  |  Pubmed ID: 26738409

Quasielastic neutron scattering (QENS) is an ideal technique for studying water transport and relaxation dynamics at pico- to nanosecond timescales and at length scales relevant to cellular dimensions. Studies of high pressure dynamic effects in live organisms are needed to understand Earth's deep biosphere and biotechnology applications. Here we applied QENS to study water transport in Shewanella oneidensis at ambient (0.1 MPa) and high (200 MPa) pressure using H/D isotopic contrast experiments for normal and perdeuterated bacteria and buffer solutions to distinguish intracellular and transmembrane processes. The results indicate that intracellular water dynamics are comparable with bulk diffusion rates in aqueous fluids at ambient conditions but a significant reduction occurs in high pressure mobility. We interpret this as due to enhanced interactions with macromolecules in the nanoconfined environment. Overall diffusion rates across the cell envelope also occur at similar rates but unexpected narrowing of the QENS signal appears between momentum transfer values Q = 0.7-1.1 Å(-1) corresponding to real space dimensions of 6-9 Å. The relaxation time increase can be explained by correlated dynamics of molecules passing through Aquaporin water transport complexes located within the inner or outer membrane structures.

Class I Hydrophobin Vmh2 Adopts Atypical Mechanisms to Self-Assemble into Functional Amyloid Fibrils

Biomacromolecules. Mar, 2016  |  Pubmed ID: 26828412

Hydrophobins are fungal proteins whose functions are mainly based on their capability to self-assemble into amphiphilic films at hydrophobic-hydrophilic interfaces (HHI). It is widely accepted that class I hydrophobins form amyloid-like structures, named rodlets, which are hundreds of nanometers long, packed into ordered lateral assemblies and do not exhibit an overall helical structure. We studied the self-assembly of the Class I hydrophobin Vmh2 from Pleurotus ostreatus in aqueous solutions by dynamic light scattering (DLS), thioflavin T (ThT), fluorescence assay, circular dichroism (CD), cryogenic trasmission electron microscopy (cryo-TEM), and TEM. Vmh2 does not form fibrillar aggregates at HHI. It exhibits spherical and fibrillar assemblies whose ratio depends on the protein concentration when freshly solubilized at pH ≥ 7. Moreover, it spontaneously self-assembles into isolated, micrometer long, and twisted amyloid fibrils, observed for the first time in fungal hydrophobins. This process is promoted by acidic pH, temperature, and Ca(2+) ions. A model of self-assembly into amyloid-like structures has been proposed.

Crossover from Localized to Diffusive Water Dynamics in Carbon Nanohorns: A Comprehensive Quasielastic Neutron-scattering Analysis

Physical Review. E. Feb, 2016  |  Pubmed ID: 26986285

Incoherent neutron scattering by water confined in carbon nanohorns was measured with the backscattering spectrometer SPHERES and analyzed in exemplary breadth and depth. Quasielastic spectra admit δ-plus-Kohlrausch fits over a wide q and T range. From the q and T dependence of fitted amplitudes and relaxation times, however, it becomes clear that the fits do not represent a uniform physical process, but that there is a crossover from localized motion at low T to diffusive α relaxation at high T. The crossover temperature of about 210 to 230 K increases with decreasing wave number, which is incompatible with a thermodynamic strong-fragile transition. Extrapolated diffusion coefficients D(T) indicate that water motion is at room temperature about 2.5 times slower than in the bulk; in the supercooled state this factor becomes smaller. At even higher temperatures, where the α spectrum is essentially flat, a few percentages of the total scattering go into a Lorentzian with a width of about 1.6μeV, probably due to functional groups on the surface of the nanohorns.

Field-induced Self-assembly of Iron Oxide Nanoparticles Investigated Using Small-angle Neutron Scattering

Nanoscale. Nov, 2016  |  Pubmed ID: 27782247

The magnetic-field-induced assembly of magnetic nanoparticles (NPs) provides a unique and flexible strategy in the design and fabrication of functional nanostructures and devices. We have investigated the field-induced self-assembly of core-shell iron oxide NPs dispersed in toluene by means of small-angle neutron scattering (SANS). The form factor of the core-shell NPs was characterized and analyzed using SANS with polarized neutrons. Large-scale aggregates of iron oxide NPs formed above 0.02 T as indicated by very-small-angle neutron scattering measurements. A three-dimensional long-range ordered superlattice of iron oxide NPs was revealed under the application of a moderate magnetic field. The crystal structure of the superlattice has been identified to be face-centred cubic.

Structure and Domain Dynamics of Human Lactoferrin in Solution and the Influence of Fe(III)-ion Ligand Binding

BMC Biophysics. 2016  |  Pubmed ID: 27822363

Human lactoferrin is an iron-binding protein of the innate immune system consisting of two connected lobes, each with a binding site located in a cleft. The clefts in each lobe undergo a hinge movement from open to close when Fe(3+) is present in the solution and can be bound. The binding mechanism was assumed to relate on thermal domain fluctuations of the cleft domains prior to binding. We used Small Angle Neutron Scattering and Neutron Spin Echo Spectroscopy to determine the lactoferrin structure and domain dynamics in solution.

Waiting
simple hit counter