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Investigating the Effect of Temperature on Transient Partial Unfolding by Proteolysis

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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.

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

Document Type: Research Article

Publication date: September 1, 2009

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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