Protocol to study CCT-mediated folding of Gβ by single-particle cryo-EM.

The chaperonin CCT mediates folding of many cytosolic proteins, including G protein β subunits (Gβs). Here, we present a protocol for isolating Gβ bound to CCT and its co-chaperone PhLP1 and determining the CCT-mediated folding trajectory of Gβ using single-particle cryoelectron microscopy (cryo-EM) techniques. We describe steps for purifying CCT-Gβ-PhLP1 from human cells, stabilizing the closed CCT conformation, preparing and imaging cryo-EM specimens, and processing data to recover multiple Gβ folding intermediates.

Visualizing the chaperone-mediated folding trajectory of the G protein β5 β-propeller.

The Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with β-propeller domains. Here, we determine the structures of human CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gβ, a component of Regulator of G protein Signaling (RGS) complexes. Cryoelectron microscopy (cryo-EM) and image processing reveal an ensemble of distinct snapshots that represent the folding trajectory of Gβ from an unfolded molten globule to a fully folded β-propeller.

Visualizing the chaperone-mediated folding trajectory of the G protein β5 β-propeller.

The cytosolic Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with β-propeller domains. Here, we determined structures of CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gβ, a component of Regulator of G protein Signaling (RGS) complexes. Cryo-EM and image processing revealed an ensemble of distinct snapshots that represent the folding trajectory of Gβ from an unfolded molten globule to a fully folded β-propeller.

Mechanistic insights into protein folding by the eukaryotic chaperonin complex CCT.

The cytosolic chaperonin CCT is indispensable to eukaryotic life, folding the cytoskeletal proteins actin and tubulin along with an estimated 10% of the remaining proteome. However, it also participates in human diseases such as cancer and viral infections, rendering it valuable as a potential therapeutic target. CCT consists of two stacked rings, each comprised of eight homologous but distinct subunits, that assists the folding of a remarkable substrate clientele that exhibits both broad diversity and specificity.

The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3.

The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca.