Design of Glyco-Linkers at Multiple Structural Levels to Modulate Protein Stability.

N-glycosylation has critical roles in regulating protein stability, but the molecular basis is poorly understood. In this study, we integrated experimental and computational techniques to investigate the mechanism by which full-length N-glycans modulate protein stability from quaternary structure perspective. We found the two inherent N-glycans of β-glucuronidase expressed in Pichia pastoris function as "glyco-linkers" that hold spatially proximal motifs together to compact the local protein structure. We further designed and placed glyco-linkers in the unusual form of glyco-bridge and glyco-hairpin at the interfaces between domains and monomers with higher structural level, respectively, which conferred dramatically higher kinetic stability and thermodynamic stability than the inherent N-glycans. Our study not only provides unique insight into the interactions between glycans and proteins from a quaternary structure perspective but also facilitates the rational design of N-glycans as general tools that can enhance protein stability.