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

Area of Science:

• Biochemistry and Structural Biology
• Mass Spectrometry Applications

Background:

• Protein structure dictates function and stability, crucial for biotechnology.
• Nonphysiological conditions can disrupt protein integrity, leading to loss of function.
• Chemical cross-linking strategies, like INCYPRO, can improve protein resistance to stress.

Purpose of the Study:

• To investigate how chemical cross-linking impacts protein structure and stability.
• To compare the structural resilience of wild-type and cross-linked Pseudomonas fluorescens Esterase (PFE).
• To evaluate the utility of nIM-MS and CIU for probing protein stability.

Main Methods:

• Utilized native ion mobility mass spectrometry (nIM-MS) for high-resolution conformational analysis.
• Employed collision-induced unfolding (CIU) to study protein unfolding pathways and resilience.
• Compared wild-type (WT) PFE with its chemically cross-linked trimeric complex.

Main Results:

• WT-PFE unfolded extensively and dissociated into monomers upon gas-phase activation.
• The cross-linked PFE complex remained compact and structurally intact under activation.
• CIU fingerprints demonstrated that cross-linked PFE resisted conformational transitions, unlike WT-PFE.

Conclusions:

• Chemical cross-linking significantly enhances protein structural integrity and stability.
• The mass spectrometry platform (nIM-MS/CIU) is effective for studying protein stability.
• Protein cross-linking shows potential for preserving native-like protein structures in challenging conditions.