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

@article{Park2005PulsePA,
  title={Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding},
  author={Chiwook Park and Susan Marqusee},
  journal={Nature Methods},
  year={2005},
  volume={2},
  pages={207-212}
}
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… 
Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis
Pulse proteolysis exploits the difference in proteolytic susceptibility between folded and unfolded proteins for facile but quantitative determination of protein stability. The method requires only
Determining protein stability in cell lysates by pulse proteolysis and Western blotting
TLDR
This work shows that thermodynamic stability of low abundant proteins can be determined reliably in cell lysates by combining pulse proteolysis with quantitative Western blotting (Pulse and Western), and demonstrates the reliability of Pulse and Western.
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An approach to observe a protein directly in a cell and to monitor a fluorescence signal that reports the unfolding transition of the protein, yielding quantitatively interpretable stability data in vivo is described.
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The results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics and determine dissociation equilibrium constant of the MBP•maltose complex from unfolding kinetic constants.
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TLDR
P pulse proteolysis is extended to determine binding affinities for a protein–protein complex involving the β‐lactamase TEM‐1 and various β‐ lactamases inhibitor protein (BLIP) mutants and accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolyses‐derived ΔCm values.
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TLDR
This work found that maltose‐binding protein is significantly stabilized by Off7, and presents an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins.
Probing membrane protein unfolding with pulse proteolysis.
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It is demonstrated that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin, which may allow the investigation of membrane protein unfolding in situ without overexpression or purification.
A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity
TLDR
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Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening.
TLDR
The data support the use of pulse proteolysis as an orthogonal, quantitative, and predictive tool to measure protein conformational stability and rank-order formulations.
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