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 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.
Highlights
From the Center for Advanced Research in Biotechnology, tUniversity ofMaryland Biotechnology Institute, and the §National Institutes of Standards and Technology, Rockville, Maryland 20850, and the Wepartment of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228
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 .1Cp of -0.85 kcal mol"! degree-I. a-Casein binds to GroEL with micromolar affinity and a positive enthalpy change in the range of 15-23°C, yielding a .1Cp of -0.44 kcal mol"! degree-I
Since a quantitative study of the interaction of molecular chaperones with nonnative polypeptides is potentially complicated by the tendency of these chains to spontaneously refold or aggregate in the absence of denaturants, an unfolded variant of subtilisin BPN' was used to measure the energetics of substrate interaction with the GroEL chaperonin from E. coli
Summary
From the Center for Advanced Research in Biotechnology, tUniversity ofMaryland Biotechnology Institute, and the §National Institutes of Standards and Technology, Rockville, Maryland 20850, and the Wepartment of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228. 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.
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