How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?

Here, we critically evaluate the effects of changes in the ion internal energy (E(int)) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the M + 6H ion of ubiquitin (ubq(6+)), the M + 5H ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the CdiMT ion. The ion-neutral CCS for ions formed by "native-state" ESI show a strong dependence on E(int). Collisional activation is used to increase E(int) prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of experimental CCSs with those generated by molecular dynamics (MD) simulation for solution-phase ions and solvent-free ions as a function of temperature provide new insights about conformational preferences and retention of solution conformations. The E(int)-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the E(int) is increased, these structurally labile conformers unfold to an elongated conformation.