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…
229 Citations
Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis
- Biology, ChemistryCurrent protocols in protein science
- 2006
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
- BiologyProtein science : a publication of the Protein Society
- 2009
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.
A method for direct measurement of protein stability in vivo.
- BiologyMethods in molecular biology
- 2009
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.
Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp
- BiologyPloS one
- 2012
A method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp), combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state is proposed.
Investigating protein unfolding kinetics by pulse proteolysis
- Biology, ChemistryProtein science : a publication of the Protein Society
- 2009
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.
Protein–protein binding affinities by pulse proteolysis: Application to TEM‐1/BLIP protein complexes
- Biology, ChemistryProtein science : a publication of the Protein Society
- 2010
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.
Quantitation of protein–protein interactions by thermal stability shift analysis
- BiologyProtein science : a publication of the Protein Society
- 2011
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.
- BiologyJournal of molecular biology
- 2011
A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity
- Biology, ChemistryProteins
- 2011
The fQCR method is demonstrated by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose‐binding protein engineered to contain single, protected cysteines, likely to find applications in protein engineering and functional genomics.
Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening.
- Medicine, ChemistryJournal of pharmaceutical sciences
- 2019
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