Vitaliy Pipich

Nucleation and Growth of CaCO3 Mediated by the Egg-White Protein Ovalbumin : A Time-Resolved in situ Study Using Small-Angle Neutron Scattering

Mineralization of calcium carbonate in aqueous solutions starting from its initiation was studied by time-resolved small-angle neutron scattering (SANS). SANS revealed that homogeneous crystallization of CaCO 3 involves an initial formation of thin plate-shaped nuclei which subsequently reassemble to 3-dimensional particles, first of fractal and finally of compact structure. The presence of the egg-white protein ovalbumin leads to a different progression of mineralization through several stages; the first step represents amorphous CaCO 3, whereas the other phases are crystalline. The formation and dissolution of the amorphous phase is accompanied by Ca (2+)-mediated unfolding and cross-linking of about 50 protein monomers showing the characteristic scattering of linear chains with a large statistical segment length. The protein complexes act as nucleation centers for the amorphous phase because of their enrichment by Ca (2+) ions. SANS revealed the sequential formation of CaCO 3 starting from the amorphous phase and the subsequent formation of the crystalline polymorphs vaterite and aragonite. This formation from less dense to more dense polymorphs follows the Ostwald-Volmer rule.

Synthesis and Characterization of Gelatin-Based Magnetic Hydrogels

A simple preparation of thermoreversible gelatin-based ferrogels in water provides a constant structure defined by the crosslinking degree for gelatin contents between 6 and 18 wt%. The possibility of varying magnetite nanoparticle concentration between 20 and 70 wt% is also reported. Simulation studies hint at the suitability of collagen to bind iron and hydroxide ions, suggesting that collagen acts as a nucleation seed to iron hydroxide aggregation, and thus the intergrowth of collagen and magnetite nanoparticles already at the precursor stage. The detailed structure of the individual ferrogel components is characterized by small-angle neutron scattering (SANS) using contrast matching. The magnetite structure characterization is supplemented by small-angle X-ray scattering and microscopy only visualizing magnetite. SANS shows an unchanged gelatin structure of average mesh size larger than the nanoparticles with respect to gel concentration while the magnetite nanoparticles size of around 10 nm seems to be limited by the gel mesh size. Swelling measurements underline that magnetite acts as additional crosslinker and therefore varying the magnetic and mechanical properties of the ferrogels. Overall, the simple and variable synthesis protocol, the cheap and easy accessibility of the components as well as the biocompatibility of the gelatin-based materials suggest them for a number of applications including actuators.

Hydration and interactions in protein solutions containing concentrated electrolytes studied by small-angle scattering

During protein crystallization and purification, proteins are commonly found in concentrated salt solutions. The exact interplay of the hydration shell, the salt ions, and protein-protein interactions under these conditions is far from being understood on a fundamental level, despite the obvious practical relevance. We have studied a model globular protein (bovine serum albumin, BSA) in concentrated salt solutions by small-angle neutron scattering (SANS). The data are also compared to previous studies using SAXS. The SANS results for dilute protein solutions give an averaged volume of BSA of 91,700 Å(3), which is about 37% smaller than that determined by SAXS. The difference in volume corresponds to the contribution of a hydration shell with a hydration level of 0.30 g g(-1) protein. The forward intensity I(0) determined from Guinier analysis is used to determine the second virial coefficient, A(2), which describes the overall protein interactions in solution. It is found that A(2) follows the reverse order of the Hofmeister series, i.e. (NH(4))(2)SO(4) < Na(2)SO(4) < NaOAc < NaCl < NaNO(3) < NaSCN. The dimensionless second virial coefficient B(2), corrected for the particle volume and molecular weight, has been calculated using different approaches, and shows that B(2) with corrections for hydration and the non-spherical shape of the protein describes the interactions better than those determined from the bare protein. SANS data are further analyzed in the full q-range using liquid theoretical approaches, which gives results consistent with the A(2) analysis and the experimental structure factor.

The low resolution structure of ApoA1 in spherical high density lipoprotein revealed by small angle neutron scattering

Spherical high density lipoprotein (sHDL), a key player in reverse cholesterol transport and the most abundant form of HDL, is associated with cardiovascular diseases. Small angle neutron scattering with contrast variation was used to determine the solution structure of protein and lipid components of reconstituted sHDL. Apolipoprotein A1, the major protein of sHDL, forms a hollow structure that cradles a central compact lipid core. Three apoA1 chains are arranged within the low resolution structure of the protein component as one of three possible global architectures: (i) a helical dimer with a hairpin (HdHp), (ii) three hairpins (3Hp), or (iii) an integrated trimer (iT) in which the three apoA1 monomers mutually associate over a portion of the sHDL surface. Cross-linking and mass spectrometry analyses help to discriminate among the three molecular models and are most consistent with the HdHp overall architecture of apoA1 within sHDL.

Equilibrium exchange kinetics in n-alkyl–PEO polymeric micelles: single exponential relaxation and chain length dependence

In this communication we present first results on the chain exchange kinetics of n-alkyl–PEO polymeric micelles by time-resolved small angle neutron scattering. We found that the rate strongly depends on the alkyl-chain length and that the relaxation function almost perfectly follows the single exponential decay predicted by theory. The key achievement of this study is the experimental verification that core block polydispersity accounts for the almost logarithmic time decay in block copolymer micelles as recently suggested by Choi et al. The results thus directly show that unimer exchange is the main mechanism for molecular exchange in block copolymer micelles.

Phase separation in semidilute aqueous poly(N-isopropylacrylamide) solutions.

The phase separation mechanism in semidilute aqueous poly(N-isopropylacrylamide) (PNIPAM) solutions is investigated with small-angle neutron scattering (SANS). The nature of the phase transition is probed in static SANS measurements and with time-dependent SANS measurements after a temperature jump. The observed critical exponents of the phase transition describing the temperature dependence of the Ornstein-Zernike amplitude and correlation length are smaller than values from mean-field theory. Time-dependent SANS measurements show that the specific surface decreases with increasing time after a temperature jump above the phase transition. Thus, the formation of additional hydrogen bonds in the collapsed state is a kinetic effect: A certain fraction of water remains as bound water in the system. Moreover, H-D exchange reactions observed in PNIPAM have to be taken into account.

KWS-2, the high intensity / wide Q-range small-angle neutron diffractometer for soft-matter and biology at FRM II

The KWS-2 small-angle neutron diffractometer operated by JCNS at FRM II is upgraded and optimized towards high intensity and wide Q-range studies of mesoscopic structures and structural changes due to rapid kinetics and becomes a dedicated SANS instrument to soft-matter and biology. The high intensity permits fast measurement of small or weak scattering samples and time resolved structural studies with a time resolution up to 100ms. The possibility to cover up to four decades in Q will soon enable structural investigation over a wide length scale, between several A and lμm. The characteristics and performance of the instrument in the conventional pinhole mode is detailed presented and the new upgrades currently in progress and aiming for boosting the instrument performance towards higher intensity and wider Q-range are reported.

Congruency between biophysical data from multiple platforms and molecular dynamics simulation of the double-super helix model of nascent high-density lipoprotein

The predicted structure and molecular trajectories from >80 ns molecular dynamics simulation of the solvated Double-Super Helix (DSH) model of nascent high-density lipoprotein (HDL) were determined and compared with experimental data on reconstituted nascent HDL obtained from multiple biophysical platforms, including small angle neutron scattering (SANS) with contrast variation, hydrogen-deuterium exchange tandem mass spectrometry (H/D-MS/MS), nuclear magnetic resonance spectroscopy (NMR), cross-linking tandem mass spectrometry (MS/MS), fluorescence resonance energy transfer (FRET), electron spin resonance spectroscopy (ESR), and electron microscopy. In general, biophysical constraints experimentally derived from the multiple platforms agree with the same quantities evaluated using the simulation trajectory. Notably, key structural features postulated for the recent DSH model of nascent HDL are retained during the simulation, including (1) the superhelical conformation of the antiparallel apolipoprotein A1 (apoA1) chains, (2) the lipid micellar-pseudolamellar organization, and (3) the solvent-exposed Solar Flare loops, proposed sites of interaction with LCAT (lecithin cholesteryl acyltransferase). Analysis of salt bridge persistence during simulation provides insights into structural features of apoA1 that forms the backbone of the lipoprotein. The combination of molecular dynamics simulation and experimental data from a broad range of biophysical platforms serves as a powerful approach to studying large macromolecular assemblies such as lipoproteins. This application to nascent HDL validates the DSH model proposed earlier and suggests new structural details of nascent HDL.

KWS-1 high-resolution small-angle neutron scattering instrument at JCNS: current state

The KWS-1 small-angle neutron scattering (SANS) instrument operated by the Julich Centre for Neutron Science (JCNS) at the research reactor FRM II of the Heinz Maier-Leibnitz Zentrum in Garching near Munich has been recently upgraded. The KWS-1 instrument was updated, from its active collimation apertures to the detector cabling. Most of the parts of the instrument were installed for the first time, including a broadband polarizer, a large-cross-section radio-frequency spin flipper, a chopper and neutron lenses. A custom-designed hexapod in the sample position allows heavy loads and precise sample positioning in the beam for conventional SANS experiments as well as for grazing-incidence SANS under applied magnetic field. With the foreseen in situ polarization analysis the main scientific topic of the instrument tends towards magnetism. The performance of the polarizer and flipper was checked with a polarized 3He cell at the sample position. The results of these checks and a comparison of test measurements on a ferrofluid in a magnetic field with polarized and nonpolarized neutrons are presented.

Monitoring the internal structure of poly(N-vinylcaprolactam) microgels with variable cross-link concentration.

The combination of a set of complementary techniques allows us to construct an unprecedented and comprehensive picture of the internal structure, temperature dependent swelling behavior, and the dependence of these properties on the cross-linker concentration of microgel particles based on N-vinylcaprolactam (VCL). The microgels were synthesized by precipitation polymerization using different amounts of cross-linking agent. Characterization was performed by small-angle neutron scattering (SANS) using two complementary neutron instruments to cover a uniquely broad Q-range with one probe. Additionally we used dynamic light scattering (DLS), atomic force microscopy (AFM), and differential scanning calorimetry (DSC). Previously obtained nuclear magnetic resonance spectroscopy (NMR) results on the same PVCL particles are utilized to round the picture off. Our study shows that both the particle radius and the cross-link density and therefore also the stiffness of the microgels rises with increasing cross-linker content. Hence, more cross-linker reduces the swelling capability distinctly. These findings are supported by SANS and AFM measurements. Independent DLS experiments also found the increase in particle size but suggest an unchanged cross-link density. The reason for the apparent contradiction is the indirect extraction of the parameters via a model in the evaluation of DLS measurements. The more direct approach in AFM by evaluating the cross section profiles of observed microgel particles gives evidence of significantly softer and more deformable particles at lower cross-linker concentrations and therefore verifies the change in cross-link density. DSC data indicate a minor but unexpected shift of the volume phase transition temperature (VPTT) to higher temperatures and exposes a more heterogeneous internal structure of the microgels with increasing cross-link density. Moreover, a change in the total energy transfer during the VPT gives evidence that the strength of hydrogen bonds is significantly affected by the cross-link density. A strong and reproducible deviation of the material density of the cross-linked microgel polymer chains toward a higher value compared to the respective linear chains has yet to be explained.