Browsing by Author "Raval, Shaunak"

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    Enabling Structural Proteomics with High Efficiency Protein Enrichment Technology
    (2023-09-13) Raval, Shaunak; Schriemer, David C.; MacCallum, Justin L.; Osthoff, Hans D.
    The functional state of proteins is inherently flexible, which allows them to interact with other biomolecules, including other proteins, to carry out many of their cellular functions. Understanding the structural dynamics of proteins and their network of associations is key to understanding their role in biology. Proteomics, the collection of mass spectrometry (MS)-based techniques to study proteins, provides a broad view of the organization of protein structure, from an individual dynamic unit to large-scale multiprotein assemblies, enabled by the application of labelling chemistries. This dissertation presents novel analytical workflows and data analysis routines to overcome current challenges in proteomics methods for the identification of protein-protein interactions (PPIs) and the study of protein conformation and dynamics. Affinity purification followed by mass spectrometry (AP-MS) is a prominent approach in the study of PPIs. However, the conventional workflow suffers from low enrichment efficiencies. I present and evaluate a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing non-specific binding. It enables the study of protein conformational analysis directly from cells as I demonstrate first by describing new concepts in data analysis for hydrogen/deuterium exchange mass spectrometry (HX-MS) and second by applying them to proteins isolated directly from cells.
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    Lactoferrin binding protein B - a bi-functional bacterial receptor protein
    (PLOS Pathogens, 2017-3-3) Ostan, Nicholas K. H.; Yu, Rong-Hua; Ng, Dixon; Lai, Christine Chieh-Lin; Pogoutse, Anastassia K.; Sharpe, Vladimir; Hepburn, Morgan; Sheff, Joey; Raval, Shaunak; Schriemer, David C.; Moraes, Trevor F.; Schryvers, Anthony B.
    Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.