Inter and intra-subunit interactions at the subunit interface of chaperonin GroEL are essential for its stability and assembly

Chaperonin GroEL helps in the folding of substrate proteins under normal and stress conditions. Although it remains stable and functional during stress conditions, the quantitative estimation of stability parameters and the specific amino-acid residues playing a role in its stability are not known in sufficient detail. The reason for poor understanding is its large size, multimeric nature, and irreversible unfolding process. The X-ray crystal structure reveals that equatorial domain forms almost all intra and inter-subunit interactions for assembly of GroEL. Considering all these facts, we adopted alternate strategies to use monomeric GroEL, native GroEL and equatorial domain mutants (GroELK4E/GroELD523K/GroELD473C) to study the assembly and stability of GroEL. Loss of inter-subunit interaction involving K4 residue of one subunit and E59, I60, E61, I62 residues of adjacent subunit due to K4E mutation affect the oligomerization efficiency of GroEL subunits while the equilibrium unfolding studies on wild-type monomeric GroEL, native GroEL, and the selected mutants together demonstrate that intra-subunit interactions involving K4 and D523 of the same subunit play a critical role in the thermodynamic stability of both native and monomeric GroEL without affecting the oligomerization of subunits. The stability order between the GroELwild-type(M) and its variants is GroELwild-type(M) ≥ GroELD473C(M)˃GroELD523K(M)˃GroELK4E.