Investigating protein unfolding kinetics by pulse proteolysis.
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 MBP*maltose complex, we have determined the dissociation equilibrium constant (K(d)) of the complex from unfolding kinetic constants, which is also in good agreement with known K(d) 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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Yu-Ran
YR
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, USA.
Park
Chiwook
C
D000465:Algorithms; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008320:Maltose; D058619:Maltose-Binding Proteins; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D015203:Reproducibility of Results; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014508:Urea
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D008320:Maltose; D014508:Urea; D016914:Ribonuclease H; D010447:Peptide Hydrolases; D013820:Thermolysin