Determining protein stability in cell lysates by pulse proteolysis and Western blotting.
Proteins require proper conformational energetics to fold and to function correctly. Despite the importance of having information on conformational energetics, the investigation of thermodynamic stability has been limited to proteins, which can be easily expressed and purified. Many biologically important proteins are not suitable for conventional biophysical investigation because of the difficulty of expression and purification. As an effort to overcome this limitation, we have developed a method to determine the thermodynamic stability of low abundant proteins in cell lysates. Previously, it was demonstrated that protein stability can be determined quantitatively by measuring the fraction of folded proteins with a pulse of proteolysis (Pulse proteolysis). Here, we show 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). To demonstrate the reliability of this method, we determined the thermodynamic stability of recombinant human H-ras added to lysates of E. coli and human Jurkat T cells. Comparison with the thermodynamic stability determined with pure H-ras revealed that Pulse and Western is a reliable way to monitor protein stability in cell lysates and the stability of H-ras is not affected by other proteins present in cell lysates. This method allows the investigation of conformational energetics of proteins in cell lysates without cloning, purification, or labeling.
Kim
Moon-Soo
MS
Department of Medicinal Chemistry and Molecular Pharmacology and Bindley Bioscience Center, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, USA.
Song
Jiao
J
Park
Chiwook
C
D015153:Blotting, Western; D004926:Escherichia coli; D006801:Humans; D019169:Jurkat Cells; D017510:Protein Folding; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013820:Thermolysin / Q000378:metabolism*
D011506:Proteins; D013820:Thermolysin; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)