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.