Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis
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- Abstract
- Table of Contents
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- Literature Cited
Abstract
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 common biochemistry and molecular biology lab equipment. Pulse proteolysis also can be used to determine the affinity of a ligand to its protein target by monitoring the change in protein stability upon ligand binding. The Basic Protocol describes the detailed procedure for determining protein stability using pulse proteolysis. For pulse proteolysis to be used for determining a protein's stability, the protein should not be digested significantly by pulse proteolysis when it is in the folded conformation. The Support Protocol describes a procedure for determining whether a protein satisfies this requirement. The principles of protein stability determination using denaturant and pulse proteolysis are also discussed.
Keywords: protein stability; proteolysis; ligand binding
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- Strategic Planning
- Basic Protocol 1: Determination of Global Protein Stability by Pulse Proteolysis
- Support Protocol 1: Verification of Resistance of Folded Proteins to the Pulse of Proteolysis
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
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Basic Protocol 1: Determination of Global Protein Stability by Pulse Proteolysis
Materials
Support Protocol 1: Verification of Resistance of Folded Proteins to the Pulse of Proteolysis
Materials
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Figure 20.11.1 Principle of pulse proteolysis. Because of the difference in proteolytic susceptibility, only unfolded protein is digested completely by pulse proteolysis. View Image -
Figure 20.11.2 Experimental design of the distribution of urea concentrations. (A ) Equilibrium unfolding of a protein with an even distribution of urea concentration for an initial survey. (B ) Equilibrium unfolding of a protein with more data points in the transition zone when an approximate C m is known. View Image -
Figure 20.11.3 Schematic diagram of procedures in the . View Image -
Figure 20.11.4 Schematic diagram of procedures in the . View Image -
Figure 20.11.5 Equilibrium unfolding of proteins by a chemical denaturant. Fraction of folded protein at each denaturant concentration is determined experimentally and then used to calculate Δ G unf (H2 O) with the linear extrapolation method (LEM). View Image -
Figure 20.11.6 Errors in C m determination from proteolysis of the folded protein populations. Only the transition zones in equilibrium unfolding curves are shown here for clarity. The apparent fraction of folded protein (solid line) is less than the actual fraction of folded protein (dashed line), which leads to a systematic error in C m determination (Δ C m ). View Image -
Figure 20.11.7 Systematic errors in C m as a function of the kinetic constant for proteolysis of the folded protein population ( k ). Δ C m is calculated by using Equation with m ‐values of −2 kcal/(mol·M) (solid line), −3 kcal/(mol·M) (long‐dashed line), and −4 kcal/(mol·M) (short‐dashed line). View Image
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Literature Cited
| Literature Cited | |
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| Myers, J.K., Pace, C.N., and Scholtz, J.M. 1995. Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4:2138‐2148. | |
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