Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements

Area of Science:

• Structural biology
• Biophysics
• Proteomics

Background:

• Protein interaction networks are crucial in structural genomics.
• Understanding the architecture of protein complexes is essential for deciphering their function.
• Existing methods may not fully capture the native structure of large protein assemblies.

Purpose of the Study:

• To present a novel ion mobility-mass spectrometry approach for characterizing protein complex subunit architecture.
• To validate the method using well-characterized protein complexes.
• To apply the method to study previously uncharacterized protein subcomplexes.

Main Methods:

• Simultaneous gas-phase measurement of mass and size for intact protein assemblies and subcomplexes.
• Utilizing ion mobility and mass spectrometry data as restraints for topological modeling.
• Formation and analysis of protein subcomplexes based on interface strength.

Main Results:

• The ion mobility-mass spectrometry approach successfully distinguished the subunit architecture of protein complexes.
• Modeled protein complexes, including ornithine carbamoyl transferase and glutamine synthetase, retained native interactions.
• Subcomplexes did not undergo significant rearrangement in solution or gas phases.

Conclusions:

• The developed ion mobility-mass spectrometry method is effective for modeling the topology of protein complexes.
• The approach preserves native protein-protein interactions, providing reliable structural insights.
• This technique offers a valuable tool for studying protein complexes, including those lacking high-resolution structures, such as human eukaryotic initiation factor 3 subcomplexes.