Investigating the Effect of Temperature on Transient Partial Unfolding by Proteolysis

Author(s): Kiwon Youn, Chiwook Park

Journal Name: Protein & Peptide Letters

Volume 16 , Issue 9 , 2009

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Protein inactivation frequently occurs through partially unfolded states under native conditions, and temperature is an important parameter that affects the susceptibility of proteins to inactivation. While the effect of temperature on global unfolding is well documented, however, experimental characterizations of the temperature effect on partial unfolding are rare. Proteolysis offers a valuable chance to investigate the temperature effect on partial unfolding. By investigating proteolysis kinetics, the energetics of the partially unfolded state responsible for proteolysis (the cleavable state) can be studied. E. coli ribonuclease H (RNase H) has been shown to be cleaved by thermolysin at the amide bond between Thr92 and Ala93 through partial unfolding. Using this cleavage as a model system, we evaluated quantitatively the temperature effect on conformational equilibrium between the native state and a cleavable state. The analysis shows that decrease in temperature from 37°C to 4°C decreases the population in the cleavable state and reduces proteolytic susceptibility of the substrate protein. The conformational change leading to the cleavable state has a temperature-independent positive ΔH° with negligible ΔCp°. This thermodynamic characteristic of partial unfolding for proteolysis is quite distinct from that of global unfolding of RNase H that has a considerable ΔCp° and a negative ΔH° at low temperature. The distinct thermodynamic characteristics of partial unfolding from global unfolding mainly result from the difference in the changes of solvent-accessible surface area, which confirms that the temperature effect on partial unfolding is strongly scaledependent.

Keywords: Proteolysis, partial unfolding, local fluctuation, stability, proteolytic susceptibility

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Article Details

Year: 2009
Page: [1093 - 1097]
Pages: 5
DOI: 10.2174/092986609789055287
Price: $65

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