Difference between revisions of "20.109(S18):Test protein activity using peptidyl-prolyl cis-trans isomerase assay (Day5)"
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FKBP12 is a class of PPIases and the activity can be measured by quantifying the isomerization and subsequent cleavage of a substrate, suc-AAFP-pNA (also written as Suc-Ala-Ala-Pro-Phe-NA). In this method, FKBP12 catalyzes the isomeration of the cis-Ala-Pro bond to a trans-Ala-Pro bond. Then a second enzyme, chymytrypsin, cleaves the trans form of the peptide. See the reaction schematic below. | FKBP12 is a class of PPIases and the activity can be measured by quantifying the isomerization and subsequent cleavage of a substrate, suc-AAFP-pNA (also written as Suc-Ala-Ala-Pro-Phe-NA). In this method, FKBP12 catalyzes the isomeration of the cis-Ala-Pro bond to a trans-Ala-Pro bond. Then a second enzyme, chymytrypsin, cleaves the trans form of the peptide. See the reaction schematic below. | ||
− | [[Image:20.109 Sp18 M2D5 PPiase.png|thumb| | + | [[Image:20.109 Sp18 M2D5 PPiase.png|thumb|600px|center]] |
When at equilibrium in solution, approximately 88% of the commercially available suc-AAFP-pNA substrate is in the trans from, leaving only a small amount of the peptide for isomerization. Because of this, the suc-AAFP-pNA substrate is prepared in trifluoroethanol (TFE) containing lithium chloride, LiCl. The Li+ ions maintain the substrate in 60% cis form. | When at equilibrium in solution, approximately 88% of the commercially available suc-AAFP-pNA substrate is in the trans from, leaving only a small amount of the peptide for isomerization. Because of this, the suc-AAFP-pNA substrate is prepared in trifluoroethanol (TFE) containing lithium chloride, LiCl. The Li+ ions maintain the substrate in 60% cis form. |
Revision as of 18:16, 3 February 2018
Contents
Introduction
Today you will test the activity of FKBP12 using a peptidyl-prolyl isomerase (PPIase) assay. PPIases catalyze cis-trans isomerization reactions that are essential to efficient protein folding in vivo. Specifically, these enzymes isomerize peptide bonds that are N-terminal to proline residues in polypeptide chains. Without PPIases, isomerization would be the rate-limiting step in protein folding.
FKBP12 is a class of PPIases and the activity can be measured by quantifying the isomerization and subsequent cleavage of a substrate, suc-AAFP-pNA (also written as Suc-Ala-Ala-Pro-Phe-NA). In this method, FKBP12 catalyzes the isomeration of the cis-Ala-Pro bond to a trans-Ala-Pro bond. Then a second enzyme, chymytrypsin, cleaves the trans form of the peptide. See the reaction schematic below.
When at equilibrium in solution, approximately 88% of the commercially available suc-AAFP-pNA substrate is in the trans from, leaving only a small amount of the peptide for isomerization. Because of this, the suc-AAFP-pNA substrate is prepared in trifluoroethanol (TFE) containing lithium chloride, LiCl. The Li+ ions maintain the substrate in 60% cis form.
Protocols
Part 1: Prepare samples for PPIase assay
Reagents
Next day: Confirm ligand binding using differential scanning fluorimetry assay