Domain insertion effectively regulates the mechanical unfolding hierarchy of elastomeric proteins: toward engineering multifunctional elastomeric proteins.

The architecture of elastomeric proteins controls fine-tuned nanomechanical properties of this class of proteins. Most elastomeric proteins are tandem modular in structure, consisting of many individually folded domains of varying stability. Upon stretching, these elements unfold sequentially following a strict hierarchical pattern determined by their mechanical stability, where the weakest element unfolds first and the strongest unfolds last. Although such a hierarchical architecture is well-suited for biological functions of elastomeric proteins, it may become incompatible with incorporating proteins of desirable functionality in order to construct multifunctional artificial elastomeric proteins, as many of these desired proteins are not evolved for mechanical purpose. Thus, exposure to a high stretching force will result in unraveling of these proteins and lead to a loss of their functionality. To overcome this challenge, we combine protein engineering with single molecule force spectroscopy to demonstrate that domain insertion is an effective strategy to control the mechanical unfolding hierarchy of multidomain proteins and effectively protect mechanically labile domains. As a proof-of-principle experiment, we spliced a mechanically labile T4 lysozyme (T4L) into a flexible loop of a mechanically stronger host domain GL5 to create a domain insertion protein. Using single molecule force spectroscopy, we showed that the mechanically labile T4L domain unfolds only after the mechanically stronger host domain GL5 has unfolded. Such a reverse mechanical unfolding hierarchy effectively protects the mechanically labile T4L domain from applied stretching force and significantly increased the lifetime of T4L. The approach demonstrated here opens the possibility to incorporate labile proteins into elastomeric proteins for engineering novel multifunctional elastomeric proteins.