Folding and unfolding pathway of chaperonin GroEL monomer and elucidation of thermodynamic parameters.

The conformation and thermodynamic stability of monomeric GroEL were studied by CD and fluorescence spectroscopy. GroEL denaturation with urea and dilution in buffer leads to formation of a folded GroEL monomer. The monomeric nature of this protein was verified by size-exclusion chromatography and native PAGE. It has a well-defined secondary and tertiary structure, folding activity (prevention of aggregation) for substrate protein and is resistant to proteolysis. Being a properly folded and reversibly refoldable, monomeric GroEL is amenable for the study of thermodynamic stability by unfolding transition methods. We present the equilibrium unfolding of monomeric GroEL as studied by urea and heat mediated unfolding processes. The urea mediated unfolding shows two transitions and a single transition in the heat mediated unfolding process. In the case of thermal unfolding, some residual structure unfolds at a higher temperature (70-75°C). The process of folding/unfolding is reversible in both cases. Analysis of folding/unfolding data provides a measure of ΔGNU H 2 O , Tm , ΔHvan and ΔSvan of monomeric GroEL. The thermodynamic stability parameter ΔGNU H 2 O is similar with both CD and intrinsic fluorescence i.e. 7.10±1.0kcal/mol. The calculated Tm , ΔHvan and ΔSvan from the thermal unfolding transition is 46±0.5°C, 43.3±0.1kcal/mol and 143.9±0.1cal/mol/k respectively.