Cyclic Ion Mobility – Collision Activation Experiments Elucidate Protein Behaviour in the Gas-Phase

<i>Elucidating the properties of intrinsically disordered proteins (IDPs) and unfolded and partially folded states of globular proteins is challenging owing to their heterogeneous and dynamic nature. Protein unfolding and misfolding is a key feature of a broad range of debilitating diseases, whilst the conformational propensities of intrinsically disordered proteins can play a significant role in modulating their activity, and the properties of unfolded states of globular proteins modulates their stability and tendency to aggregate. Ion mobility-mass spectrometry (IM-MS) is a powerful method for interrogating these systems, however limits in resolution and the difficulty in probing the energetics of interconversions amongst heterogeneous ensembles are major issues. Herein, using a quadrupole/cyclic-IM/ time-of-flight MS instrument, we show how the combination of precursor mass selection, mobility selection (IMⁿ) and collisional activation (CA) allows the elucidation of complicated gas-phase dynamic behavior. The methodology employed is general and is demonstrated using a classic model globular protein, cytochrome C, and an aggregation-prone IDP, amylin. CA allows investigations of protein conformational dynamics and unfolding in the gas-phase for heterogeneous mixtures, whilst the additional precursor mass selection capability provides high resolution and selectivity, facilitating more in-depth investigation. Understanding protein dynamics in the gas-phase will allow greater insight into protein behaviour and allow application of gas-phase techniques to clinically relevant systems. </i>

Cyclic Ion Mobility – Collision Activation Experiments Elucidate Protein Behaviour in the Gas-Phase

<i>Elucidating the properties of intrinsically disordered proteins (IDPs) and unfolded and partially folded states of globular proteins is challenging owing to their heterogeneous and dynamic nature. Protein unfolding and misfolding is a key feature of a broad range of debilitating diseases, whilst the conformational propensities of intrinsically disordered proteins can play a significant role in modulating their activity, and the properties of unfolded states of globular proteins modulates their stability and tendency to aggregate. Ion mobility-mass spectrometry (IM-MS) is a powerful method for interrogating these systems, however limits in resolution and the difficulty in probing the energetics of interconversions amongst heterogeneous ensembles are major issues. Herein, using a quadrupole/cyclic-IM/ time-of-flight MS instrument, we show how the combination of precursor mass selection, mobility selection (IMⁿ) and collisional activation (CA) allows the elucidation of complicated gas-phase dynamic behavior. The methodology employed is general and is demonstrated using a classic model globular protein, cytochrome C, and an aggregation-prone IDP, amylin. CA allows investigations of protein conformational dynamics and unfolding in the gas-phase for heterogeneous mixtures, whilst the additional precursor mass selection capability provides high resolution and selectivity, facilitating more in-depth investigation. Understanding protein dynamics in the gas-phase will allow greater insight into protein behaviour and allow application of gas-phase techniques to clinically relevant systems. </i>

Topological constraints and modular structure in the folding and functional motions of GlpG, an intramembrane protease

We investigate the folding of GlpG, an intramembrane protease, using perfectly funneled structure-based models that implicitly account for the absence or presence of the membrane. These two models are used to describe, respectively, folding in detergent micelles and folding within a bilayer, which effectively constrains GlpG's topology in unfolded and partially folded states. Structural free-energy landscape analysis shows that although the presence of multiple folding pathways is an intrinsic property of GlpG’s modular functional architecture, the large entropic cost of organizing helical bundles in the absence of the constraining bilayer leads to pathways that backtrack (i.e., local unfolding of previously folded substructures is required when moving from the unfolded to the folded state along the minimum free-energy pathway). This backtracking explains the experimental observation of thermodynamically destabilizing mutations that accelerate GlpG’s folding in detergent micelles. In contrast, backtracking is absent from the model when folding is constrained within a bilayer, the environment in which GlpG has evolved to fold. We also characterize a near-native state with a highly mobile transmembrane helix 5 (TM5) that is significantly populated under folding conditions when GlpG is embedded in a bilayer. Unbinding of TM5 from the rest of the structure exposes GlpG’s active site, consistent with studies of the catalytic mechanism of GlpG that suggest that TM5 serves as a substrate gate to the active site.