Robotically assisted titration coupled to ion mobility-mass spectrometry reveals the interface structures and...

Area of Science:

• Biochemistry and structural biology
• Macromolecular complex analysis
• Protein structure and dynamics

Background:

• Assessing macromolecular structures is crucial for understanding biochemistry and disease.
• Current methods for structural analysis lag behind functional identification efforts.

Purpose of the Study:

• To develop and apply a high-throughput method for probing protein complex structures.
• To investigate the impact of solution conditions on protein-protein interfaces.
• To correlate complex disruption with interface characteristics.

Main Methods:

• Coupling ion mobility-mass spectrometry (IM-MS) with automated robotic sampling.
• Systematic variation of solvent compositions (ionic strength and DMSO content).
• Analysis of eight different protein homotetramers.

Main Results:

• Altering ionic strength and DMSO content disrupted all studied protein-protein interfaces.
• Ion mobility measurements of intact assemblies and subcomplexes generally matched X-ray structures.
• Distorted subcomplexes were identified under extreme conditions, allowing for data correction.
• Strong correlations were found between complex disruption intensity and interface interaction types.

Conclusions:

• The combined IM-MS and robotic sampling approach effectively probes protein complex stability and interfaces.
• This method can quantify protein interfacial interactions in unknown proteins after calibration.
• The findings advance structural biology techniques for disease-related macromolecular studies.