From Solution to Gas Phase: Revealing Ligand-Dependent Confor-mations of Ribonuclease A with Tandem-Trapped Ion Mobility Spectrometry

Protein activity depends on motional transitions between conformational states. Modifications or ligand binding can alter protein dynamics, leading to changes in activity. Established biophysical methods effectively determine protein structures but face challenges when investigating dynamic biological processes that involve steady states of transiently populated con-formations. Ion mobility/mass spectrometry shows promise for studying transient protein conformations but characterizes them in a solvent-free environment and does not directly provide detailed structural information. Here, we investigate to what extent subtly different conformational states are retained in their solvent-free environment. To this end, we investigate the conformations of liganded and unliganded ribonuclease A (RNase A) using our tandem-trapped ion mobility spectrome-ter/tandem-mass spectrometer (Tandem-TIMS) in conjunction with molecular dynamics-based computational approaches. RNase A transitions between a closed and an open conformation on the millisecond timescale, with the closed conformation being favored when liganded. Our results indicate that both ligand-bound and unliganded RNase A maintain approximately 80% of their native contacts in a solvent-free environment. This includes crucial interactions between the ligand and the protein scaffold, as well as the ligand's position within the binding pocket. Furthermore, our analysis reveals that when RNase A is ligand-bound, it adopts a more compact structure, as observed in solution. Additionally, significant differences between the open and closed conformations—such as the positioning of Loop 1—are mostly preserved in the solvent-free state, with root mean squared deviations of about 2 Å. In summary, our findings demonstrate the ability of Tandem-TIMS to characterize subtle structural differences between steady-state protein conformations. This ability assumes increased sig-nificance due to the crucial involvement of transient protein assemblies in cellular signaling and neurodegenerative diseases, as well as “hidden” protein states underlying enzyme function that are not directly accessible using established methods.