Metastability of Protein Solution Structures in the Absence of Solvent: Rugged Energy Landscape and Glass-Like Behavior

Despite the significance of differentially modified proteins (proteoforms) to human health, it remains challenging to identify how proteoforms alter protein structural dynamics and function. Although native ion mobility/mass spectrometry is well-suited to handle proteoform heterogeneity, it characterizes protein structures in the absence of solvent. This raises long-standing, unanswered questions about the biological significance of structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the structural denaturation of the protein ubiquitin in the solvent-free environment. We show that ubiquitin exists in the absence of solvent as an ensemble of kinetically stable subpopulations that are separated by substantial free energy barriers. These subpopulations unfold but do not interconvert, which indicates that the solvent-free subpopulations originate from different solution-phase conformations. The subpopulations exhibit stretched-exponential denaturation kinetics consistent with a glass transition associated with separating the C-terminal beta-strand from the N-terminal beta-hairpin occurring on the folded side of the unfolding transition state. Our data indicate that this transition state is highly polarized with significant native content in the N-terminal beta-hairpin and alpha-helix, resembling the transition state reported for the presence of a solvent. Taken together, our analysis suggests that ubiquitin in the solvent-free environment reflects the conformational ensemble of ubiquitin in solution because the initially formed solvent-free state of ubiquitin appears glass-like and “melts” over several seconds.