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

Miri Sokolovski, Jelena Cveticanin, Déborah Hayoun, Ilia Korobko, Michal Sharon, Amnon Horovitz

The strength and specificity of protein complex formation is crucial for most life processes and is determined by interactions between residues in the binding partners. Double-mutant cycle analysis provides a strategy for studying the energetic coupling between amino acids at the interfaces of such complexes. Here we show that these pairwise interaction energies can be determined from a single high-resolution native mass spectrum by measuring the intensities of the complexes formed by the two wild-type proteins, the complex of each wild-type protein with a mutant protein, and the complex of the two mutant proteins. This native mass spectrometry approach, which obviates the need for error-prone measurements of binding constants, can provide information regarding multiple interactions in a single spectrum much like nuclear Overhauser effects (NOEs) in nuclear magnetic resonance. Importantly, our results show that specific inter-protein contacts in solution are maintained in the gas phase.Double mutant cycle (DMC) analyses can provide the interaction energies between amino acids at the interface of protein complexes. Here, the authors determine pairwise interaction energies using high-resolution native mass spectroscopy, offering a straightforward route for the DMC methodology.