An Engineered Recombinant Spider Silk Protein Providing Disulfide-Locked Control over Self-Assembly and Fiber Formation

Abstract

Spider silks are renowned for their mechanical properties. The notably tough Argiope trifasciata aciniform silk is spun from a protein predominantly comprising a series of identical 200 residue units (“W units”). In solution, each W unit has a globular 5-helix domain connected to its neighbors by intrinsically disordered linkers while the fiber contains mixed α-helical, β-sheet and disordered structuring. Helix 5 is more dynamic and prone to denaturation than the remainder of the globular domain, implying that it may structurally transform during fiber spinning. Rational cysteine substitutions are introduced at a proximal pair of serines in helix 5 and in helix 1 of the globular domain. Upon disulfide formation, the helix 5 C-terminal region loses helicity and experiences increased backbone dynamics, while the remainder of the globular structure is effectively unperturbed. Fiber formation by hand-drawing and wet-spinning is prevented in the disulfide-locked state, but readily possible in the reduced (“unlocked”) state. The disulfide-locked state differs from the unlocked state with much more heterogeneous pre-fibrillar assembly in spinning dope and through prevention of β-sheet formation upon cooling following thermal denaturation. This engineered protein thus provides a chemically-reversible disulfide-locked state of aciniform silk with modified self-assembly propensity and fiber formation.