Abstract

This dissertation addresses key bottlenecks in protein X-ray crystallography by developing a simplified purification strategy based on reversible protein self-assembly and applying it to the structural characterization of the therapeutically important kinase VPS34. The first objective of this study was to develop a simplified, chromatography-independent protein purification strategy by exploiting the reversible pH-dependent polymerization of the Translocation ETS Leukemia Sterile Alpha Motif (TELSAM) crystallization chaperone. Repeated cycles of polymerization and resolubilization enabled the direct purification and concentration of TELSAM-fusion proteins from bacterial lysates, eliminating several conventional purification steps. Validation using three previously characterized TELSAM-fusion proteins demonstrated that compatible targets could be purified to crystallization-grade quality and yielded crystal structures indistinguishable from those obtained using traditional chromatographic methods. The results further showed that the success of this approach depends largely on the biochemical stability of the fused protein during repeated pH cycling, defining both its utility and limitations as a rapid protein purification strategy. The second objective focused on the structural characterization of VPS34, the sole class III phosphoinositide 3-kinase in humans and an important therapeutic target implicated in cancer, neurodegenerative disorders, and infectious diseases. This study describes the expression, purification, crystallization, and high-resolution structural determination of the VPS34 catalytic domain, following optimization of protein production and crystallization conditions to obtain homogeneous protein and diffraction-quality crystals. Crystal structures of VPS34 in complex with the selective inhibitor RD-I-53 and five analogues were determined to elucidate the structural basis of inhibitor binding. Comparative analyses revealed that modifications at RD-I-53's benzothiazole C6 position alter ligand orientation, stability, and water-mediated interactions within the ATP-binding pocket, thereby influencing binding affinity. Comparison with computational docking predictions further demonstrated that uncommon receptor side-chain conformations can lead to inaccurate ligand pose predictions, highlighting the continued importance of experimental structural determination for validating computational models and guiding structure-based inhibitor design.

Degree

PhD

College and Department

Computational, Mathematical, and Physical Sciences; Chemistry and Biochemistry

Rights

https://lib.byu.edu/about/copyright/

Date Submitted

2026-08-14

Document Type

Dissertation

Keywords

TELSAM, VPS34, PIK3C3, protein purification, protein crystallization, structure-guided drug design, kinase inhibitors, ligand binding, autophagy, pH-dependent polymerization, structure-activity relationship, molecular docking, protein engineering

Language

english

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