Measuring inter-protein pairwise interaction energies from a single native mass spectrum by double-mutant cycle...

Area of Science:

• Biochemistry and Molecular Biology
• Biophysical Chemistry
• Structural Biology

Background:

• Protein complex formation is vital for biological processes, driven by specific residue interactions at binding interfaces.
• Double-mutant cycle (DMC) analysis is a key strategy for quantifying energetic coupling between amino acids in protein complexes.
• Traditional DMC analysis often relies on measuring binding constants, which can be prone to errors and require extensive experimentation.

Purpose of the Study:

• To develop a streamlined method for determining pairwise interaction energies at protein-protein interfaces.
• To leverage high-resolution native mass spectrometry for efficient double-mutant cycle analysis.
• To validate that inter-protein contacts observed in solution are preserved in the gas phase during mass spectrometry.

Main Methods:

• Utilized high-resolution native mass spectrometry to analyze protein complexes.
• Measured the relative intensities of complexes formed by wild-type and mutant proteins (homo- and hetero-dimers).
• Calculated pairwise interaction energies directly from mass spectrometry data, bypassing binding constant measurements.

Main Results:

• Demonstrated that pairwise interaction energies can be accurately determined from a single native mass spectrum.
• Showcased the ability to analyze multiple inter-residue interactions simultaneously within one experiment.
• Confirmed the conservation of specific inter-protein contacts in the gas phase compared to solution.

Conclusions:

• Native mass spectrometry offers a direct and robust approach for performing double-mutant cycle analysis.
• This method simplifies the study of protein complex energetics, providing insights comparable to NMR techniques like NOEs.
• The findings establish a powerful new tool for investigating the molecular basis of protein-protein interactions.