No abstract

In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer ionic strength, the formation of the gel network around unfolding conditions prevents investigations of protein aggregation. In this study, by deploying the fact that in lysozyme solutions the time of folding/unfolding is much shorter than the characteristic time of gelation, we have prevented gelation by rapidly heating the solution up to the unfolding temperature (~80 °C) for a short time (~30 min.) followed by fast cooling to the room temperature. Dynamic light scattering measurements show that if the gelation is prevented, nanosized irreversible aggregates (about 10–15 nm radius) form over a time scale of 10 days. These small aggregates persist and aggregate further into larger aggregates over several weeks. If gelation is not prevented, the nanosized aggregates become the building blocks for the gel network and define its mesh length scale. These results support our previously published conclusion on the nature of mesoscopic aggregates commonly observed in solutions of lysozyme, namely that aggregates do not form from lysozyme monomers in their native folded state. Only with the emergence of a small fraction of unfolded proteins molecules will the aggregates start to appear and grow.

Formation of inhomogeneous (in the form of a “coffee ring”) or homogeneous deposits accompanies the drying of a particle-laden drop. Invariably, this deposition occurs in a two-dimensional (2D) space (x, y plane) (and might have a finite thickness in z), where the evaporating drop is positioned. Here, we show an interesting extension of this problem: we demonstrate the occurrence of evaporation-mediated particle deposits that span three dimensions (x, y, and z). The extent of the span in this 3rd dimension (z) is comparable to the span in x and y and hence is much larger than the finite thickness (in z) of the 2D deposits. Particle-laden drops are introduced in an uncured and heavier (than the drop) polydimethysiloxane (PDMS) film, enabling the drop to come to the uncured PDMS surface and breach it and get partly exposed to the surrounding air enforcing the onset of evaporation. The subsequent curing of the drop-laden PDMS film ensures that the drop is occupying a three-dimensional (3D) cavity; as a consequence, the evaporation-driven flow field, depending on the particle sizes, leads to a deposition pattern that spans three dimensions. We consider particles of three different sizes: coffee particles (20–50 μm), silver nanoparticles (∼20 nm), and carbon nanotubes (CNTs) (1–2 μm). The coffee particles form a ring-like deposit in the x, y plane, while the much smaller silver nanoparticles (NPs) and CNTs form a 3D deposit that spans in x, y, and z directions. We anticipate that the present finding of the evaporation-triggered three-dimensional (3D) particle deposits will enable unprecedented self-assembly-driven fabrication of various materials, structures, and functional devices as well as patterning and coating in 3D spaces.

Cells assemble dynamic protein-based nanostructures far from equilibrium, such as microtubules, in a process referred to as dissipative assembly. Synthetic analogues have utilized chemical fuels and reaction networks to form transient hydrogels and molecular assemblies from small molecule or synthetic polymer building blocks. Here, we demonstrate dissipative cross-linking of transient protein hydrogels using a redox cycle, which exhibit protein unfolding-dependent lifetimes and mechanical properties. Fast oxidation of cysteine groups on bovine serum albumin by hydrogen peroxide, the chemical fuel, formed transient hydrogels with disulfide bond cross-links that degraded over hours by a slow reductive back reaction. Interestingly, despite increased cross-linking, the hydrogel lifetime decreased as a function of increasing denaturant concentration. Experiments showed that the solvent-accessible cysteine concentration increased with increasing denaturant concentration due to unfolding of secondary structures. The increased cysteine concentration consumed more fuel, which led to less direction oxidation of the reducing agent and affected a shorter hydrogel lifetime. Increased hydrogel stiffness, disulfide cross-linking density, and decreased oxidation of redox-sensitive fluorescent probes at a high denaturant concentration provided evidence supporting the unveiling of additional cysteine cross-linking sites and more rapid consumption of hydrogen peroxide at higher denaturant concentrations. Taken together, the results indicate that the protein secondary structure mediated the transient hydrogel lifetime and mechanical properties by mediating the redox reactions, a feature unique to biomacromolecules that exhibit a higher order structure. While prior works have focused on the effects of the fuel concentration on dissipative assembly of non-biological molecules, this work demonstrates that the protein structure, even in nearly fully denatured proteins, can exert similar control over reaction kinetics, lifetime, and resulting mechanical properties of transient hydrogels.

No abstract