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Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding

Nature Methods volume 2, pages 207–212 (2005)Cite this article

Abstract

Thermodynamic stability is fundamental to the biology of proteins. Information on protein stability is essential for studying protein structure and folding and can also be used indirectly to monitor protein-ligand or protein-protein interactions. While clearly valuable, the experimental determination of a protein's stability typically requires biophysical instrumentation and substantial quantities of purified protein, which has limited the use of this technique as a general laboratory method. We report here a simple new method for determining protein stability by using pulse proteolysis with varying concentrations of denaturant. Pulse proteolysis is designed to digest only the unfolded proteins in an equilibrium mixture of folded and unfolded proteins that relaxes on a time scale longer than the proteolytic pulse. We used this method to study the stabilities of Escherichia coli ribonuclease H and its variants, both in purified form and directly from cell lysates. The ΔGunf° values obtained by this technique were in agreement with those determined by traditional methods. We also successfully used this method to monitor the binding of maltose-binding protein to maltose, as well as to rapidly screen cognate ligands for this protein. The simplicity of pulse proteolysis suggests that it is an excellent strategy for the high-throughput determination of protein stability in protein engineering and drug discovery applications.

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Figure 1: Principle of pulse proteolysis.
Figure 2: Effect of urea concentration on intrinsic proteolysis rate.
Figure 3: Determination of Cm of RNase H* by pulse proteolysis.
Figure 4: Monitoring ligand association by pulse proteolysis.

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Acknowledgements

We thank J.A. Blair for technical help with the cloning of MBP and the Bustamante lab for the use of their Typhoon imaging system. This work was supported by National Institutes of Health grant GM50945.

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  1. Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley, Berkeley, 94720, California, USA

    Chiwook Park & Susan Marqusee

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  1. Chiwook Park
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Correspondence to Susan Marqusee.

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The authors declare no competing financial interests.

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Park, C., Marqusee, S. Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding. Nat Methods 2, 207–212 (2005). https://doi.org/10.1038/nmeth740

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