Controllable coacervation of recombinantly produced spider silk protein using kosmotropic salts

Bioinspired functionally graded composite assembled using cellulose nanocrystals and genetically engineered proteins with controlled biomineralization

“…Nanocomposite assembly of impact resistant interior region of the crown Previously we described how a diverse set of structural proteins can undergo ion-induced liquid-liquid phase separation (LLPS) in vitro, resulting in the spontaneous formation of two immiscible liquid phases from the dispersed protein solution by loss of solvation. 21,25,30,31 In the present study, LLPS of the reinforcing proteins was initiated by mixing a solution of 1 M potassium phosphate at pH 7.4 with 100 μM protein, resulting in a clear dilute phase on top and a dense coacervate phase on the bottom of the vial with droplet sizes in the range of 0.05 -1 μm (Figure 2g-f and S2-6). To assemble nanocomposite lms, we intermixed CNC (3 % w/v) with the RPs in their coacervated form (10 % with respect to the dry mass of CNC).…”

“…First, we set out to explore the use of reinforcing structural proteins to produce a tough fracture energy-dissipating matrix (Figure 2d and Figure S1). [20][21][22][23] We hypothesized that such proteins would enable bridging interactions between the stiff long-range chiral nematic helicoidal CNC scaffold, thus acting as a molecular binder. Second, our earlier reports demonstrate that to use these proteins as a binding matrix for biocomposites, it is crucial to initially induce their phase separation and condensation into highly concentrated assemblies, often described as coacervates (Figure 2e-f and S2-S6).…”

“…[24][25][26][27] Key advantages of concentrated coacervates include increased intermolecular interactions, low viscosity and interfacial tension, all of which greatly facilitate wetting and in ltration through the CNC network to result in strong binding to the reinforcing phase and, in turn, to high adhesive properties that could not be achieved in the absence of coacervation (Figure 2h). [20][21][22][23] Furthermore, we have recently found that CMP-1 in the club of the peacock mantis shrimp has a 3-block primary structure (Figure 2d) 14 that may result in all three of the characteristics required to act as a matrix of a tough biocomposite. First, it has a conserved mid-block carbohydrate-binding module that is likely used as an anchor to chitin brils in the club.…”

“…Such dramatic differences in properties can be attributed to the lack of poly-Ala stretches in ELP's core sequence. Earlier observations have shown that Ala-rich sequences undergo conformational conversion from random coil/α-helical to a β-sheet dominated content, 20,21 which increases proteinprotein interactions and results in a more cohesive matrix with more e cient load transfer. Such a molecular network creates interlocking regions that dissipate the mechanical stress between the protein chains and have been found to signi cantly toughen natural silk (Figure S6).…”

Interfacial crystallization and supramolecular self-assembly of spider silk inspired protein at the water-air interface

“…Their tri-block architecture is based on our earlier reports consisting of two folded terminal domains and a highly repetitive amphiphilic midblock spidroin sequence (Figure 1). [20][21][22][23] We anticipated the geometry of the pendant droplet interface to be the most suitable approach for understanding the self-assembly and conformational evolution of dense silk solution with concentrations of spidroins in the same range as in the glands before ber formation. This is otherwise problematic to probe using any other means such as planar surfaces as described previously using very dilute protein solutions.…”