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Suppressing the influence of humidity in protein-based materials is central to their use in a variety of applications. It is believed that protein charge plays a key role in water uptake. Therefore, in this work, whey protein was neutralized, supercharged, and superneutralized to examine the effects of protein modification on moisture absorption in protein copolymers. The charge-modified proteins were formulated into thermoset elastomers through a three-step process: methacrylation, complexation with various surfactants, and co-polymerization with n-butyl acrylate. Compatibility of the protein and hydrophobic acrylate monomer can be tuned through changes in surfactant type, ratio between surfactant and protein, and protein charge modification. Using benzalkonium chloride as the surfactant compatibilizer, elastomers with the various modified proteins were prepared using a melt polymerization approach. Acetylation and esterification of whey protein, which neutralize charged functional groups, resulted in the reduction of the proteins' water uptake relative to unmodified whey. Once incoporated into elastomers, all copolymers regardless of protein modifications have similar moisture contents. However, elastomers with superneutralized proteins demonstrated a lowered mechanical dependence on humidity, presented as a smaller change in elongation at break and tensile strength compared to a copolymer based on non-charge modified whey. This journal is

Protein-polymer bioconjugate self-assembly has attracted a great deal of attention as a method to fabricate protein nanomaterials in solution and the solid state. To identify protein properties that affect phase behavior in protein-polymer block copolymers, a library of 15 unique protein-b-poly(N-isopropylacrylamide) (PNIPAM) copolymers comprising 11 different proteins was compiled and analyzed. Many attributes of phase behavior are found to be similar among all studied bioconjugates regardless of protein properties, such as formation of micellar phases at high temperature and low concentration, lamellar ordering with increasing temperature, and disordering at high concentration, but several key protein-dependent trends are also observed. In particular, hexagonal phases are only observed for proteins within the molar mass range 20-36 kDa, where ordering quality is also significantly enhanced. While ordering is generally found to improve with increasing molecular weight outside of this range, most large bioconjugates exhibited weaker than predicted assembly, which is attributed to chain entanglement with increasing polymer molecular weight. Additionally, order-disorder transition boundaries are found to be largely uncorrelated to protein size and quality of ordering. However, the primary finding is that bioconjugate ordering can be accurately predicted using only protein molecular weight and percentage of residues contained within β sheets. This model provides a basis for designing protein-PNIPAM bioconjugates that exhibit well-defined self-assembly and a modeling framework that can generalize to other bioconjugate chemistries., United States. Department of Energy. Office of Basic Energy Sciences (Award DE-SC0007106)

Charge anisotropy or the presence of charge patches at protein surfaces has long been thought to shift the coacervation curves of proteins and has been used to explain the ability of some proteins to coacervate on the "wrong side" of their isoelectric point. This work makes use of a panel of engineered superfolder green fluorescent protein mutants with varying surface charge distributions but equivalent net charge and a suite of strong and weak polyelectrolytes to explore this concept. A patchiness parameter, which assessed the charge correlation between points on the surface of the protein, was used to quantify the patchiness of the designed mutants. Complexation between the polyelectrolytes and proteins showed that the mutant with the largest patchiness parameter was the most likely to form complexes, while the smallest was the least likely to do so. The patchiness parameter was found to correlate well with the phase behavior of the protein-polymer mixtures, where both macrophase separation and the formation of soluble aggregates were promoted by increasing the patchiness depending on the polyelectrolyte with which the protein was mixed. Increasing total charge and increasing strength of the polyelectrolyte promote interactions for oppositely charged polyelectrolytes, while charge regulation is also key to interactions for similarly charged polyelectrolytes, which must interact selectively with oppositely charged patches.