Tyrosine Mutation in the Characteristic Motif of the Amorphous Region of Spidroin for Self-Assembly Capability Enhancement

Spidroin, with robust mechanical performance and good biocompatibility, could fulfill broad applications in material science and biomedical fields. Development of miniature spidroin has made abundant fiber production economically feasible, but the mechanical properties of artificial silk still fall short of natural silk. The mechanism behind mechanical properties of spidroin usually focuses on beta-microcrystalline regions; the effect of amorphous regions was barely studied. In this study, residue tyrosines (Y) were designed to replace asparagine (N)/glutamic acid (Q) in the characteristic motifs (GGX)n in amorphous regions for performance enhancement of spidroin; the mutants presented lower free energy and significantly exhibited stronger van der Waals and electrostatic interactions, which might result from pi-pi stacking interactions between the phenyl rings in the side chain of tyrosine. Additionally, the soluble expressions of wild-type spidroin and mutant spidroin were achieved when heterologously expressed in E. coli, with yields of 560 mg/L (2REP), 590 mg/L (2REPM), 240 mg/L (4REP), and 280 mg/L (4REPM). Significantly, secondary structure analysis confirmed that the mutant spidroin more avidly forms more beta-sheets than the wild-type spidroin, and aggregation morphology suggested that mutant spidroin displayed better self-assembly capacity and was easier to form artificial spider silk fibers; in particular, self-assembled 4REPM nanofibrils had an average modulus of 11.2 +/- 0.35 GPa, about 2 times higher than self-assembled B. mori silk nanofibrils and almost the same as that of native spider dragline silk fibers (10-15 GPa). Thus, we first demonstrated a new influence mechanism of the amorphous region's characteristic motif on the self-assembly and material properties of spidroin. Our study provides a reference for the design of high-performance material proteins and their heterologous preparation.