Stabilizing Proteins by Chemical Cross-Linking: Insights into Conformation, Unfolding, and Aggregation Using Native...

The function and stability of proteins depend on their three-dimensional structure, which includes conformational dynamics and potential self-assembly. Protein structural organization is particularly important in biotechnological applications, where protein integrity is often challenged by nonphysiological conditions, leading to disassembly, aggregation, and eventually the loss of function or activity. The use of chemical cross-linking strategies, such as the in situ cyclization of proteins (INCYPRO), can overcome these challenges, providing proteins and protein complexes with enhanced resistance to thermal and chemical stress. To probe how cross-linking affects protein structure and stability, we combined native ion mobility mass spectrometry (nIM-MS) and collision-induced unfolding (CIU). Here, we compare the wild-type (WT) and chemically cross-linked trimeric complex of Pseudomonas fluorescens Esterase (PFE), using nIM-MS to obtain high-resolution insights into the conformations, while CIU allowed the investigation of unfolding pathways and structural resilience under activation. We show that upon gas-phase activation, the native enzyme undergoes extensive unfolding and dissociation into monomers, whereas the cross-linked form remains compact and structurally intact. CIU fingerprints show that, when combining tunnel-in pressure and DC voltages, WT-PFE undergoes extensive structural unfolding, whereas the cross-linked complex resists conformational transitions. This mass spectrometry platform offers a powerful approach to study protein stability and, in this case, highlights the potential of protein cross-linking for preserving native-like structures.