Protocol to study CCT-mediated folding of Gβ₅ by single-particle cryo-EM

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The chaperonin containing TCP-1 (CCT) complex is essential for folding numerous cytosolic proteins.
• G protein β subunits (Gβs) are crucial cytosolic proteins that require CCT for proper folding.
• Understanding the folding mechanisms of Gβs by CCT is vital for cellular function.

Purpose of the Study:

• To present a detailed protocol for isolating G protein β5 (Gβ5) bound to CCT and its co-chaperone PhLP1.
• To determine the folding trajectory of Gβ5 mediated by CCT using advanced imaging techniques.
• To enable visualization of the chaperonin-mediated protein folding process.

Main Methods:

• Isolation and purification of the CCT-Gβ5-PhLP1 complex from human cells.
• Stabilization of the closed conformation of the CCT complex.
• Single-particle cryo-electron microscopy (cryo-EM) for specimen preparation, imaging, and data processing.

Main Results:

• Successful isolation of the CCT-Gβ5-PhLP1 complex.
• Recovery of multiple Gβ5 folding intermediates through cryo-EM data analysis.
• Visualization of distinct stages in the CCT-mediated folding pathway of Gβ5.

Conclusions:

• The developed protocol allows for the visualization of protein folding mediated by the chaperonin CCT.
• This method provides insights into the dynamic process of Gβ5 folding.
• The study offers a valuable tool for investigating chaperonin-assisted protein maturation.