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Jandu, Rupinder

University of British Columbia

Membrane proteins (MPs) are central to numerous cellular processes and represent key targets in drug discovery, yet their characterization can be challenging due to their hydrophobic nature and reliance on detergents for solubilization. Traditional workflows may destabilize MPs, hindering the study of their ligand interactions and downstream effects. In this thesis, we develop a novel membrane mimetic-based thermal proteome profiling (MM-TPP) workflow that aims to overcome these challenges by enabling the investigation of ligand-specific interactions on membrane proteomes in a detergent-free environment. Using peptidisc, we successfully capture integral membrane proteins (IMPs) into water-soluble libraries, facilitating downstream thermal proteome profiling. Using ATP and vanadate together (ATP-VO₄) as ligands, we validate the approach in both bacterial cells and mouse organ. Our results highlight ATP-VO₄-mediated stabilization of IMPs such as the bacterial ABC transporters MsbA and LolCD. As well as liver transporters like FATP2/SLC27A2 and BSEP/ABCB11. Beyond small-molecule ligands, MM-TPP highlights the effects of co-captured lipids on protein stability, underscoring the broader scope of this methodology. Additionally, MM-TPP has the potential to detect off-target stabilization effects of ATP derivatives, such as ADP, on non-ATP-binding proteins. showcasing its ability to uncover ligand metabolism and downstream interactions. This thesis establishes an introduction to MM-TPP as a basis for membrane proteome characterization.

Text

2025-04-10

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/90626

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/