High-density chemical cross-linking for modeling protein interactions.

Area of Science:

• Biochemistry
• Structural Biology
• Proteomics

Background:

• Understanding protein complex 3D structure is crucial for mechanistic insights.
• Traditional structure elucidation methods are costly, labor-intensive, and require pure samples.
• Chemical cross-linking coupled with mass spectrometry (MS) is an emerging alternative, often combined with cryo-electron microscopy.

Purpose of the Study:

• To develop an improved chemical cross-linking and mass spectrometry method for high-resolution protein complex structural analysis.
• To demonstrate the method's capability in modeling protein-protein interactions and large molecular machines.
• To provide a scaffold for developing novel protein-based inhibitors.

Main Methods:

• Integration of orthogonal cross-linking chemistries.
• Advancements in search algorithms, statistical analysis, and computational efficiency.
• Application to carbonic anhydrase-inhibitor complex and the yeast proteasome.

Main Results:

• Achieved coverage of 1 unique cross-linked position pair per 7 amino acids at a 1% false discovery rate without peptide fractionation.
• Successfully modeled the carbonic anhydrase-inhibitor interaction interface at high resolution.
• Identified 3,893 unique cross-linked peptides for the yeast proteasome, enabling de novo modeling of its regulatory particle.

Conclusions:

• The developed method significantly enhances protein complex structural understanding.
• Cross-linking data reflects conformational dynamics and disorder in large complexes.
• The method's high cross-linking density is sufficient for de novo structural modeling of complex assemblies.