The Hydrophobic Nature of GroEL-Substrate Binding

Abstract

The molecular chaperone GroEL from Escherichia coli is a member of the highly conserved Hsp60 family of proteins that facilitates protein folding. A central question regarding the mechanism of GroEL-assisted refolding of proteins concerns its broad substrate specificity. The nature of GroEL-polypeptide chain interaction was investigated by isothermal titration calorimetry using proteins that maintain a non-native conformation in neutral buffer solutions. A single molecule of an unfolded variant of subtilisin BPN' binds non-cooperatively to GroEL with micromolar affinity and a positive enthalpy change. Additional calorimetric titrations of this chain with GroEL show that the positive enthalpy change decreases with increasing temperature between 6 and 25°C, yielding a ΔCp of −0.85 kcal mol -1 degree -1. α-Casein similarly binds to GroEL with micromolar affinity and a positive enthalpy change in the range of 15-23°C, yielding a ΔCp of −0.44 kcal mol -1 degree -1. The negative heat capacity change provides strong evidence for the role of hydrophobic interactions as the driving force for the association of these substrates with the GroEL chaperonin. The molecular chaperone GroEL from Escherichia coli is a member of the highly conserved Hsp60 family of proteins that facilitates protein folding. A central question regarding the mechanism of GroEL-assisted refolding of proteins concerns its broad substrate specificity. The nature of GroEL-polypeptide chain interaction was investigated by isothermal titration calorimetry using proteins that maintain a non-native conformation in neutral buffer solutions. A single molecule of an unfolded variant of subtilisin BPN' binds non-cooperatively to GroEL with micromolar affinity and a positive enthalpy change. Additional calorimetric titrations of this chain with GroEL show that the positive enthalpy change decreases with increasing temperature between 6 and 25°C, yielding a ΔCp of −0.85 kcal mol -1 degree -1. α-Casein similarly binds to GroEL with micromolar affinity and a positive enthalpy change in the range of 15-23°C, yielding a ΔCp of −0.44 kcal mol -1 degree -1. The negative heat capacity change provides strong evidence for the role of hydrophobic interactions as the driving force for the association of these substrates with the GroEL chaperonin.