Comparing the Conformational Stability of Pyruvate Kinase in the Gas Phase and in Solution.

Collision induced unfolding (CIU) is increasingly used to characterize protein complexes in the gas phase and is often employed to detect ligand binding-induced conformational stabilization. However, the extent to which gas-phase conformational stabilities measured by CIU reflect analogous parameters in solution is not yet clear, particularly for systems where conformational and protein complex stability are modulated by point mutation. Here, we compare CIU-derived relative stabilities of four point mutants of the homotetramer pyruvate kinase to solution stabilities measured by differential scanning fluorimetry (DSF) and solution conformational dynamics measured by time-resolved electrospray ionization hydrogen-deuterium exchange (TRESI-HDX). Our results demonstrate that both destabilization of the tetrameric state and generally reduced conformational stability of the monomer in solution are well correlated to lower onset energies for specific unfolding transitions observed in CIU. However, this correlation not fully retained when comparing CIU to HDX data, where the latter measurement is strongly impacted by conformational dynamics within the tetramer.