Coacervation of resilin fusion proteins containing terminal functionalities

Abstract

Liquid-liquid phase transition known as coacervation of resilin-like-peptide fusion proteins containing different terminal domains were investigated. Two different modular proteins were designed and produced and their behavior were compared to a resilin-like-peptide without terminal domains. The size of the particle-like coacervates was modulated by the protein concentration, pH and temperature. The morphology and three-dimensional (3D) structural details of the coacervate particles were investigated by cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET) reconstruction. Selective adhesion of the coacervates on cellulose and graphene surfaces was demonstrated.

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

“…Consequently, we recorded 1 H→ 31 P heteronuclear correlation (HETCOR) two dimensional (2D) magic-angle spin nuclear magnetic resonance (MAS-NMR), with the expectation that this would enable selective enhancement of protonated species in close proximity with phosphate ions of crystalline apatite mineral (Figure 4c and Figure S19). The 16-day sample showed a relatively sharp 31 P signal at 1.57 ppm correlated with OH -1 at δ ( 1 H) at around 0.0 ppm corresponding to ordered PO 4 3of the apatite crystallite as previously described. 35,36 In all other samples, the corresponding 31 P/ 1 H cross-peak was broader, indicating lower crystallinity (Figure S20).…”

“…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).…”

Resilin-mimetics as a smart biomaterial platform for biomedical applications

“…Furthermore, in order to study the effect of different terminal domain on coacervate size and structure, two modular RLPs—namely CBM 2 -RLP-HFBI and CBM-RLP-CBM—comprising cellulose-binding module (CBM) and amphiphilic hydrophobin protein domain (HFBI) were synthesized and investigated 49 . The salt-induced coacervate size of CBM 2 -RLP-HFBI was observed to be larger than CBM-RLP-CBM (observed using cryo-TEM) due to hydrophobic interactions of HFBI, and tuneable with varying protein concentration, temperature and pH 49 . The modular RLPs developed so far have great potential for controlled release, drug delivery, biosensor and injectable hydrogel applications.…”

“…Spherical to cylindrical morphology transition with increase in RLP chain length and hydrophobicity of the ELP Drug delivery, tissue engineering 48 CBM 2 -RLP-HFBI, CBM-RLP-CBM Cellulose-binding module (CBM), hydrophobin protein domain (HFBI) Increase in coacervate size with increase in temperature and pH. Selective adhesion and self-assembly on different surfaces Tissue engineering, drug delivery, biosensor 49 , 56 GB1-RLP 4 , GB1-RLP-(GB1) 5 -RLP-(GB1) 4 -RLP Artificial elastomeric protein (GB1) Increase in mechanical property Tissue engineering 66 RLP 12 -LCD 5 Lysine crosslinking domain (LCD) Increase in lysine crosslinking site and mechanical property Tissue engineering 75 a The RLPs, namely An X and RZ 10 are constructed using the mosquito BX619161 gene, whereas the rest using the fruit fly CG15920 gene. …”

“…one-pot approach) [53][54][55] . These FMNCs are composed of a Lately, advancement in directed self-assembly of modular RLP-based systems was performed using CBM 2 -RLP-HFBI and CBM-RLP-CBM, where dip-coated CBM 2 -RLP-HFBI coacervate adhered as non-continuous layer to both cellulose and graphene surfaces, whereas CBM-RLP-CBM coacervate adhered as separate micro-sized particles to only cellulose surface (observed using AFM) 49 . In addition, mechanical extension experiments performed at different pH (5 and 11) using CBM 2 -RLP-HFBI by adhering CBM 2 end to a thin layer of cellulose on one side and HFBI end to octadecyl trichlorosilane (OTS)-coated AFM tip on the other side showed directed or force-induced conformational stretching of the RLP chain 56 .…”