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

Protein activity depends on motional transitions between conformational states. Modifications or ligand binding can alter the 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 conformations. 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. We investigate the conformations of unliganded ribonuclease A (RNase A) and RNase A bound to uracil-3'-monophosphate (3'-UMP) and adenosine-5'-monophosphate (5'-AMP) using our tandem-trapped ion mobility spectrometer/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 time scale, with the closed conformation being favored when liganded. A comparison of our experimental and computational results indicates that both ligand-bound and unliganded RNase A maintain approximately 80% of their native contacts in a solvent-free environment. This includes interactions between the ligand and the protein, as well as the ligand's position within the binding pocket. Furthermore, our experimental data reveal that when solvent-free RNase A is ligand-bound, it maintains a more compact structure, as observed in solution. Additionally, differences between the open and closed conformationssuch as the positioning of Loop 1are mostly preserved in the solvent-free state per our computational analysis, with root-mean-square 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 takes on increased significance due to the 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.