The full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period, even for Texas A&M users with NetID.
Evaluating Subunit Interfacial Interactions and Chloride Adduction Induced Gas-Phase Stabilization of Diubiquitin using ESI-IM-MS and Collisional Activation Methods
MetadataShow full item record
Native electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) allows for the investigation of complex heterogeneous mixtures of intact biomolecules according to their mass, charge, and size, which can provide unique insight into higher order protein structure (i.e. tertiary, quaternary, etc.), solution-phase perturbations, and dynamics. However, this method is more of a “coarse-grained” approach providing relative size information rather than more detailed characterization of local structures. Here we demonstrate the use of the complementary technique collision induced unfolding (CIU) for the characterization of noncovalent intra/intermolecular interactions of the model protein ubiquitin (Ubq), its bioactive oligomers, and the effect of chloride adduction on conformational preference. Ubq is a relatively small protein that participates in a wide-array of bioactivities as defined by the ability to form a variety of oligomeric states. A key facet to the quaternary structure and thus function of Ubq oligomers is the site of covalent-linkage between the subunits. Here we present the investigation of the recently reported non-covalent homodimer of Ubq (ncUbq) and four diubiquitin (diUbq) molecules covalently-linked through different lysine side-chains. Despite that each covalent-linkage site induces distinct conformational preferences owing to steric hindrance induced by the covalent tether, ESI-IM-MS lacks sufficient resolution to differentiate these conformers; however, the CIU fingerprints of each are distinctly different. Furthermore, the CIU fingerprint of ncUbq suggests a high degree of conformational similarity to diUbq covalently linked through K48. ESI-IM-MS is an inherently gas-phase technique, raising the question: How much does the solvent-free ion resemble the solution-phase structure of the analyte, and under what conditions might this structure be preserved? Here, using ESI-IM-MS and CIU, we provide evidence that chloride adduction to Ubq results in gas-phase stabilization of a more compact conformer; furthermore, the more compact conformers of chloride adducted K48-linked diUbq ions demonstrate gas-phase unfolding patterns characteristic of the “native-like” structure reported in the previous sections. Thus we present evidence that chloride adduction to Ubq and K48-linked diUbq ions results in the gas-phase stabilization of the “native-like” conformation of the analyte ion and inducing additional resistance to gas-phase unfolding.
Collision Induced Unfolding
Wagner, Nicole DeAnne (2017). Evaluating Subunit Interfacial Interactions and Chloride Adduction Induced Gas-Phase Stabilization of Diubiquitin using ESI-IM-MS and Collisional Activation Methods. Doctoral dissertation, Texas A & M University. Available electronically from