The hydrophobic nature of GroEL-substrate binding.

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 degrees C, yielding a delta CP of -0.85 kcal mol-1 degree-1. alpha-Casein similarly binds to GroEL with micromolar affinity and a positive enthalpy change in the range of 15-20 degrees C, yielding a delta CP 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.