Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding.
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 DeltaG(unf) degrees 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.
Park
Chiwook
C
Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley, Berkeley, California 94720, USA.
Marqusee
Susan
S
D004355:Drug Stability; D004789:Enzyme Activation; D004795:Enzyme Stability; D017022:Flow Injection Analysis / Q000379:methods*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods*; D016914:Ribonuclease H / Q000032:analysis* / Q000737:chemistry*; D013820:Thermolysin / Q000737:chemistry
D016914:Ribonuclease H; C078856:ribonuclease HI; D010447:Peptide Hydrolases; D013820:Thermolysin