Abstract

All organisms depend on cellular signaling pathways to dictate cell growth, responses to stimuli, and all other cellular functions necessary to maintain health and survival. Many of these signals rely on G protein coupled receptors (GPCRs) and their associated heterotrimeric G proteins for signal detection and transduction. To perform this function, the three subunits of the G protein must be assembled after synthesis on the ribosome. This process requires additional proteins called molecular chaperones to assist in folding of the subunits and assembly into the G protein heterotrimer. Among these chaperones, the cytosolic chaperonin CCT plays an essential role in this process. CCT is an essential protein folding machine with a diverse clientele of substrates, including many proteins with WD40-repeat sequences that fold into β-propeller domains, such as those belonging to the G protein β subunit family (Gβ). The Gβ family is comprised of Gβ subunits 1-4 that form obligate with Gγ subunits that then associate with Gα subunits to form G protein heterotrimers. In contrast, the fifth member of the Gβ family (Gβ5) forms obligate dimers with the R7 family of regulators of G protein signaling (RGS) proteins that set the duration of cellular signals transduced by GPCRs throughout the central nervous system. To understand how the subunits of these vital signaling proteins are folded and assembled into their respective complexes, we have used cryo-electron microscopy, image processing and model refinement, and biochemical methods to determine how CCT and its co chaperone phosducin-like protein 1 (PhLP1) interact with and fold members of the Gβ family. We first solved the folding trajectory of Gβ5, which showed a complete transition from a molten globule state to the fully folded β-propeller. These structures answered a long-standing question of how CCT accommodates diverse clients with unique folding requirements and dispels the notion that CCT passively folds its substrates. Instead, we demonstrate that CCT actively participates in folding through specific contacts to facilitate the sequential folding of individual β-sheets until the propeller closes into its native structure. We also present a detailed protocol for isolating Gβ5 bound to CCT and PhLP1 and determining the CCT-mediated folding trajectory of Gβ5 using single-particle cryo-EM techniques. To better understand the impact of mutations on the Gβ5 folding trajectory, we used biochemistry and cryo-EM to determine how missense mutations in Gβ5, including those that cause severe neurological diseases, alter the Gβ5 folding trajectory and lead to incompletely folded, trapped intermediates. Finally, we investigated the CCT-mediated folding of Gβ1, another member of the Gβ subunit family, revealing that CCT directs Gβ1 folding through initiating specific intermolecular contacts distinct from those made with Gβ5.

Degree

PhD

College and Department

Computational, Mathematical, and Physical Sciences; Chemistry and Biochemistry

Rights

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