Characterization of group II chaperonins from an acidothermophilic archaeon Picrophilus torridus

Abstract

Chaperonins are a type of molecular chaperone that assist in the folding of proteins. Group II chaperonins play an important role in the proteostasis in the cytosol of archaea and eukarya. In this study, we expressed, purified, and characterized group II chaperonins from an acidothermophilic archaeon Picrophilus torridus . Two genes exist for group II chaperonins, and both of the gene products assemble to form double‐ring complexes similar to other archaeal group II chaperonins. One of the Picrophilus chaperonins, Pto CPN α, was able to refold denatured GFP at 50 °C. As expected, Pto CPN α exhibited an ATP ‐dependent conformational change that is observed by the change in fluorescence and diffracted X‐ray tracking ( DXT ). In contrast, Pto CPN α lost its protein folding ability at moderate temperatures, becoming unable to interact with unfolded proteins. At lower temperatures, the release rate of the captured GFP from Pto CPN α was accelerated, and the affinity of denatured protein to Pto CPN α was weakened at the lower temperatures. Unexpectedly, in the DXT experiment, the fine motions were enhanced at the lower temperatures. Taken together, the results suggest that the fine tilting motions of the apical domain might correlate with the affinity of group II chaperonins for denatured proteins.

Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC

“…The various CtCCT variants were constructed for further research. Previously, we analyzed the ATP-dependent motion of TKS1-Cpn at the single molecule level by DXT [ 10 , 21 , 22 ]. Gold nanocrystals were used as tracers for the subunit motion of CtCCT.…”

“…The conformational transition induces the folding of the encapsulated protein. Finally, group II chaperonins reopen to release the folded protein from the cavity [ 10 , 11 ]. Group II chaperonins cooperate with a co-chaperone called prefoldin.…”

Dynamic 3D visualization of active protein′s motion using diffracted X-ray tracking

“…Tilting motion of the nanocrystal reflects in the diffraction spot movement in a radial direction in time-resolved diffraction images In the usual setting, the angular resolution is one mrad (0.057°) in tilting direction and five mrad (0.286°) in twisting direction. This method is sensitive in rotational motions and applied for motion analysis of several proteins, including membrane proteins, 16,17) multimer proteins, 18,19) de novo designed peptide 20) and rubbers. 21) The diffraction spot from the nanocrystal is observed when the angular position of nanocrystal that satisfy the Bragg diffraction condition (2d sin θ = n λ, d: lattice spacing of the nanocrystals θ: scattering angle, λ: wavelength of incident X-ray, and n: integer).…”

Diffracted X-ray Tracking for Observing the Internal Motions of Individual Protein Molecules and Its Extended Methodologies

“…As shown in Figure 9, DXT is applicable to a variety of proteins [32][33][34][35][36][37][38]. From these many DXT measurements, labeling larger gold nanocrystals slightly reduces the measured kinetic size, as shown in Figure 10 The surface of the deposited, fabricated gold nanocrystals is water-repellent.…”