Structural Evolution of Metastable Protein Aggregates in the Presence of Trivalent Salt Studied by (V)SANS and SAXS

We present a study of the structural evolution of protein aggregates formed in solutions of a globular protein, β-lactoglobulin (BLG), in the presence of YCl₃. These aggregates are often observed before crystallization starts and they are metastable with respect to the crystalline phase. Here we focus on the characterization of the hierarchical structure of this intermediate phase and its temperature dependent structure evolution using a combination of (very) small angle neutron and X-ray scattering (VSANS, SANS, and SAXS). Results show that the hierarchical structure ranges from nanometer scale protein monomer, dimer and compact protein clusters to micrometer scale fractal protein aggregates. Upon cooling, the overall hierarchical structure is preserved, but the evolution of the internal structure within the aggregates is clearly visible: the monomer–monomer correlation peak reduces its intensity and disappears completely at lower temperatures, whereas the cluster–cluster correlation is enhanced. At a larger length scale, the fractal dimension of protein aggregates increases. The kinetics of the structure change during a temperature ramp was further investigated using time-resolved SAXS. The time dependent SAXS profiles show clear isosbestic points and the kinetics of the structural evolution can be well described using a two-state model. These dynamic properties of protein aggregates on a broad length scale may be essential for being the precursors of nucleation

Effects of Biological Molecules on Calcium Mineral Formation Associated with Wastewater Desalination as Assessed using Small-Angle Neutron Scattering

Calcium phosphate scale formation on reverse osmosis (RO) membranes is one of the main limitations on cost-effective desalination of domestic wastewater worldwide. It has been shown that organic agents affect mineralization. In this study, we explored mineralization in the presence of two biofilm-relevant organic compounds, the proteins bovine serum albumin (BSA) and lysozyme, in a simulated secondary effluent (SSE) solution using small-angle neutron scattering (SANS), and applied the results to analyses of mineral precipitation in RO desalination of secondary effluents of wastewater. The two proteins are prominent members of bacterial extracellular polymeric substances (EPSs), forming biofilms that are frequently associated with RO-membrane fouling during wastewater desalination. Laboratory experiments showed that both proteins in SSE solution are involved in complex mineralization processes. Only small portions of both protein fractions are involved in mineralization processes, whereas most of the protein fractions remain as monomers in solution. Contrast variation showed that composite particles of mineral and protein are formed instantaneously to a radius of gyration of about 300 Å, coexisting with particles of about μm size. After about one day, these large particles start to grow again at the expense of the 300 Å particles. The volume fraction of the 300 Å particles is of the order of 2 × 10^–4, which is too large to represent calcium phosphate such as hydroxyapatite as the only mineral present. Considering the data of mineral volume fraction obtained here as well as the solubility product of possible mineral polymorphs in the SSE solution, we suggest the formation of protein-mineral particles of hydroxyapatite and calcium carbonate during scale formation

Monitoring the Internal Structure of Poly(<i>N</i>‑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 <i>N</i>-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

Pressure-Dependence of Poly(<i>N</i>‑isopropylacrylamide) Mesoglobule Formation in Aqueous Solution

Above their cloud point, aqueous solutions of the thermoresponsive polymer poly­(<i>N</i>-isopropylacrylamide) (PNIPAM) form large mesoglobules. We have carried out very small-angle neutron scattering (VSANS with <i>q</i> = 0.21–2.3 × 10^–3 Å^–1) and Raman spectroscopy experiments on a 3 wt % PNIPAM solution in D₂O at atmospheric and elevated pressures (up to 113 MPa). Raman spectroscopy reveals that, at high pressure, the polymer is less dehydrated upon crossing the cloud point. VSANS shows that the mesoglobules are significantly larger and contain more D₂O than at atmospheric pressure. We conclude that the size of the mesoglobules and thus their growth process are closely related to the hydration state of PNIPAM