NDLI: Investigating protein unfolding kinetics by pulse proteolysis

Content Provider	PubMed Central
Author	Na, Yu-ran Park, Chiwook
Copyright Year	2009
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 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.
Related Links	http://dx.doi.org/10.1002/pro.29
Ending Page	276
Page Count	9
Starting Page	268
File Format	PDF
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science : A Publication of the Protein Society
Issue Number	2
Volume Number	18
Language	English
Publisher	Wiley Subscription Services, Inc., A Wiley Company
Publisher Date	2009-02-01
Access Restriction	Open
Rights Holder	Wiley Subscription Services, Inc., A Wiley Company
Subject Keyword	Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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