Protocol to study CCT-mediated folding of Gß5 by single-particle cryo-EM.

The chaperonin CCT mediates folding of many cytosolic proteins, including G protein ß subunits (Gßs). Here, we present a protocol for isolating Gß5 bound to CCT and its co-chaperone PhLP1 and determining the CCT-mediated folding trajectory of Gß5 using single-particle cryoelectron microscopy (cryo-EM) techniques. We describe steps for purifying CCT-Gß5-PhLP1 from human cells, stabilizing the closed CCT conformation, preparing and imaging cryo-EM specimens, and processing data to recover multiple Gß5 folding intermediates. This protocol permits visualization of protein folding by CCT. For complete details on the use and execution of this protocol, please refer to Sass et al.1.

Visualizing the chaperone-mediated folding trajectory of the G protein ß5 ß-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 ß-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 Gß5, 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 Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT orchestrates folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.

Visualizing the chaperone-mediated folding trajectory of the G protein ß5 ß-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 ß-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 Gß5, 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 Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß-sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT directs folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.