Efficient and Controllable Assembly of Multitype Polyubiquitin Chains via Enzyme-Assisted Thiol Ligation for Structural Elucidation

Methods for constructing polyubiquitin chains have greatly advanced our understanding of their structures, dynamics, and cellular functions. However, efficiently assembling multiple ubiquitin (Ub) units into well-defined chains with controlled lengths and distinct linkages under physiological conditions, while preserving the native three-dimensional structures of target proteins, remains a challenge. Addressing these issues would facilitate high-resolution characterization of dynamics and interactions of polyubiquitin chains involved in physiological processes using biophysical techniques. Herein, we report a versatile strategy for constructing multitype polyubiquitin chains via orthogonal ligation of expressed Ub units by combining 5-fluoro-4-(phenylsulfonyl)pyridine-2-carbonitrile (FPPN)-mediated consecutive protein–protein coupling with transulfurase-catalyzed protection and deprotection of the C-terminal cysteine. Both the thiol cross-ligation and the enzymatic protection–deprotection reactions rapidly proceed under physiological conditions with high conversion efficiency, enabling directional and controllable assembly of polyubiquitin chains. The approach allows the preparation of various homogeneous and mixed polyubiquitin chains with defined linkages and lengths, as well as branched polyubiquitin chains. Construction of polyubiquitin chains containing isotopically enriched Ub units and/or site-directed spin-labeled conjugates further demonstrates that this strategy permits high-resolution elucidation of polyubiquitin-chain conformations by NMR and EPR in solution and in situ, revealing that the conformational ensemble of K11/K48-branched Ub 3 shifts toward a more compact state in the cellular environment. This work presents a robust and broadly applicable platform for constructing diverse ubiquitin chains and ubiquitinated proteins, facilitating in situ high-resolution characterization of polyubiquitin-associated structures, dynamics, and interactions.