Modulating Hierarchical Self-Assembly In Thermoresponsive Intrinsically Disordered Proteins Through High-Temperature Incubation Time

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium. Here, we report that high-temperature incubation time is a defining parameter for micro and nanoscale self-assembly of resilin-like IDPs. Interestingly, high-resolution scanning electron microscopy micrographs reveal that an extended incubation time leads to the formation of micron-size rods and ellipsoids that depend on the amino acid sequence. More surprisingly, a prolonged incubation time also induces amino acid composition-dependent formation of short-range nanoscale order, such as periodic lamellar nanostructures. We can correlate the lamellar structures to \b{eta}-sheet formation and demonstrate similarities between the observed nanoscopic structural arrangement and spider silk. We, therefore, suggest that regulating the period of high-temperature incubation, in the one-phase regime, can serve as a unique method of controlling the hierarchical self-assembly mechanism of structurally disordered proteins.Comment: 27pages, 8 figure

Activating hidden signals by mimicking cryptic sites in a synthetic extracellular matrix

Abstract Cryptic sites are short signaling peptides buried within the native extracellular matrix (ECM). Enzymatic cleavage of an ECM protein reveals these hidden peptide sequences, which interact with surface receptors to control cell behavior. Materials that mimic this dynamic interplay between cells and their surroundings via cryptic sites could enable application of this endogenous signaling phenomenon in synthetic ECM hydrogels. We demonstrate that depsipeptides (“switch peptides”) can undergo enzyme-triggered changes in their primary sequence, with proof-of-principle studies showing how trypsin-triggered primary sequence rearrangement forms the bioadhesive pentapeptide YIGSR. We then engineered cryptic site-mimetic synthetic ECM hydrogels that experienced a cell-initiated gain of bioactivity. Responding to the endothelial cell surface enzyme aminopeptidase N, the inert matrix transformed into an adhesive synthetic ECM capable of supporting endothelial cell growth. This modular system enables dynamic reciprocity in synthetic ECMs, reproducing the natural symbiosis between cells and their matrix through inclusion of tunable hidden signals

Surfactant-Mediated Co-Existence of Single-Walled Carbon Nanotube Networks and Cellulose Nanocrystal Mesophases

Hybrids comprising cellulose nanocrystals (CNCs) and percolated networks of single-walled carbon nanotubes (SWNTs) may serve for the casting of hybrid materials with improved optical, mechanical, electrical, and thermal properties. However, CNC-dispersed SWNTs are depleted from the chiral nematic (N*) phase and enrich the isotropic phase. Herein, we report that SWNTs dispersed by non-ionic surfactant or triblock copolymers are incorporated within the surfactant-mediated CNC mesophases. Small-angle X-ray measurements indicate that the nanostructure of the hybrid phases is only slightly modified by the presence of the surfactants, and the chiral nature of the N* phase is preserved. Cryo-TEM and Raman spectroscopy show that SWNTs networks with typical mesh size from hundreds of nanometers to microns are distributed equally between the two phases. We suggest that the adsorption of the surfactants or polymers mediates the interfacial interaction between the CNCs and SWNTs, enhancing the formation of co-existing meso-structures in the hybrid phases

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility

raw data for all the characterization and activity experiments presented in the manuscript. Including: SAXS, CD, blots, cell assay, MS et