Single-Molecule Force Spectroscopy Study on Modular Resilin Fusion Protein

Abstract

The adhesive and mechanical properties of a modular fusion protein consisting of two different types of binding units linked together via a flexible resilin-like-polypeptide domain are quantified. The adhesive domains have been constructed from fungal cellulose-binding modules (CBMs) and an amphiphilic hydrophobin HFBI. This study is carried out by single-molecule force spectroscopy, which enables stretching of single molecules. The fusion proteins are designed to self-assemble on the cellulose surface, leading into the submonolayer of proteins having the HFBI pointing away from the surface. A hydrophobic atomic force microscopy (AFM) tip can be employed for contacting and lifting the single fusion protein from the HFBI-functionalized terminus by the hydrophobic interaction between the tip surface and the hydrophobic patch of the HFBI. The work of rupture, contour length at rupture and the adhesion forces of the amphiphilic end domains are evaluated under aqueous environment at different pHs.

Coacervation of resilin fusion proteins containing terminal functionalities

“…This observation relates to our earlier study of the dCBM-RLP-HFBI molecule where the forces related to the adhesion of the hydrophobin motif was studied by single molecule force spectroscopy. [17] At elevated pH, the strength of the hydrophobic interaction of the molecules was significantly higher compared to the forces measured at the IEP. [17] The reason for the large hydrophobic interaction of the dCBM-RLP-HFBI molecules was likely a consequence of the bundling of the extended resilin domains, which led to the formation of a larger hydrophobic patch as several hydrophobins were brought closely together.…”

“…[17] At elevated pH, the strength of the hydrophobic interaction of the molecules was significantly higher compared to the forces measured at the IEP. [17] The reason for the large hydrophobic interaction of the dCBM-RLP-HFBI molecules was likely a consequence of the bundling of the extended resilin domains, which led to the formation of a larger hydrophobic patch as several hydrophobins were brought closely together. Thus, the coacervate cluster formation could be due to hydrophobic patches formed on the coacervate surface.…”

“…Synthetic Drosophila melanogaster Rec1-resilin fusion proteins were expressed in Trichoderma reesei. The production and purification of the proteins have been described earlier; RLP, CBM-RLP-CBM [16] and dCBM-RLP-HFBI [17].…”

Resilin-mimetics as a smart biomaterial platform for biomedical 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. …”

“…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 . A single molecular stretching of 6% was observed for the modular RLP at pH 5 with random coil conformation, which could not be fully extended by tensile stress, whereas 11% stretching was observed at pH 11 with swollen coil conformation, which could be fully extended by tensile stress 56 . Such directed responsiveness and self-assembly of RLPs have potential to find applications in biosensing and soft-robotics.…”