Article

Investigating protein unfolding kinetics by pulse proteolysis

Yu-Ran Na 1 2, Chiwook Park 1 2 *

1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907 2Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907

email: Chiwook Park (chiwook@purdue.edu)

^*Correspondence to Chiwook Park, 575 Stadium Mall Drive, Purdue University, West Lafayette, IN 47907

Keywords

pulse proteolysis • unfolding kinetics • maltose binding protein • ribonuclease H • ligand binding • dissociation equilibrium constant • folding • stability

Abstract

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed pulse proteolysis to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBPmaltose complex, we have determined the dissociation equilibrium constant (Kd) of the complex from unfolding kinetic constants, which is also in good agreement with known Kd values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.

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Received: 22 September 2008; Revised: 31 October 2008; Accepted: 4 November 2008

Digital Object Identifier (DOI)

10.1002/pro.29  About DOI