Abstract
Cyclization is a promising strategy to enhance protein stability, but its applicability is often limited by structural constraints such as the distance between the N- and C-terminal regions. Here, we report the rational design and characterization of a cyclized Tobacco Etch Virus protease (cTEVp) using the SpyRing system, which enables covalent cyclization through SpyTag/SpyCatcher-mediated isopeptide bond formation. We applied this approach to a widely used engineered TEVp variant (L56V, S135G, S219V, Δ238-242) and employed AlphaFold structure prediction to optimize linker length and domain positioning. Despite the ∼40 Å separation between the N- and C-termini of native TEVp, AlphaFold modeling suggested that the fused SpyTag and SpyCatcher domains can adopt a favorable configuration for intramolecular cyclization. The resulting cTEVp exhibited proteolytic activity comparable to the noncyclized TEVp, indicating that structural constraint via SpyRing-mediated cyclization did not impair enzymatic function. Importantly, cTEVp displayed significantly improved thermostability relative to its noncyclized counterpart, as demonstrated by higher retention of soluble enzyme and residual activity following heat treatment at 50 °C. Our findings validate the effectiveness of SpyRing-mediated cyclization in improving TEVp stability and highlight the utility of computationally guided cyclization as a generalizable strategy for engineering thermally resilient proteins. This study establishes a framework that integrates structure prediction and rational protein engineering, contributing to the development of robust biocatalysts for synthetic biology and industrial biotechnology applications.