Difference between revisions of "20.109(S18):Test protein activity using peptidyl-prolyl cis-trans isomerase assay (Day5)"

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.

Upon cleavage by chymotrypsin, the release of p-nitroaniline results in a yellow color in alkaline conditions that can be measured at 405 nm. The rate of Δ405 nm in the presence versus absence of FKBP12 is used to calculate the activity.

Protocols

Part 1: Prepare samples for PPIase assay

1. Calculate dilutions for assay buffer preparation.
   • Calculate the volume of assay buffer required for 18 reactions (you will need buffer for 15 reactions total, but it is best to prepare extra to account for pipetting errors) given that each reaction requires 178 μL.
   • Calculate the volume of 1 M HCl, pH = 8 needed for a final concentration of 200 mM.
   • Calculate the volume of 2 μM chymotrypsin needed for a final concentration of 20 nM.
   • Confirm your math with the teaching faculty before proceeding.
2. Obtain aliquots of 1 M HCl, pH = 8, 2 μM chymptrypsin, and sterile water from the front laboratory bench.
3. Prepare your assay buffer using the calculations completed in Step #1, then chill on ice.
4. Each team will setup triplicate reactions for 5 different conditions:
   • Condition 1: assay buffer with suc-AAFP-pNA substrate
   • Condition 2: assay buffer with suc-AAFP-pNA substrate AND FKBP12 protein
   • Condition 3: assay buffer with suc-AAFP-pNA substrate AND FKBP12 protein AND rapamycin
   • Condition 4: assay buffer with suc-AAFP-pNA substrate AND FKBP12 protein AND ligand #1
   • Condition 5: assay buffer with suc-AAFP-pNA substrate AND FKBP12 protein AND ligand #2
5. Label 5 1.5 mL centrifuge tubes, one for each condition.
6. Add assay buffer to each tube for 3.25 reactions, again it is best to prepare extra for pipetting error.
7. Calculate the volume of DMSO needed for a final concentration of 0.02% (volume:volume) using the final reaction volume of 200 μL.
   • Because DMSO is used to dissolve rapamycin and the ligands, it must be included in all reactions to reduce unintended variables into the experiment.
   • Add the appropriate volume of DMSO to the tubes for Condition 1 and Condition 2.
8. Calculate the volume of Z μM rapamycin needed for a final concentration of 10 μM using the final reaction volume of 200 μM.
   • Add the appropriate volume of rapamycin to the tube for Condition 3.
9. Calculate the volume of each ligand (confirm stock concentration with teaching faculty) needed for a final concentration of 40 μM using the final reaction volume of 200 μL.
10. Alert the teaching faculty when your master mixes are ready.
    • When told to do so, transfer 180 μL of each master mix to three wells of a 96-well plate. Multiple teams will share each plate. Be mindful that you are adding your samples to the appropriate wells according to the plate map provided by the teaching faculty.

After all teams have loaded their samples, the teaching faculty will add 20 μL of the suc-AAFP-pNA substrate (5 mM in TFE containing 460 mM LiCl) to the samples and immediately load the plate onto the plate reader. The plate reader will record the 405 nm values at 25 °C for each sample every minute for 30 minutes (a total of 31 readings including t = 0).

Part 2: Calculate activity of FKBP12

The data for your PPIase experiment will be provided as an excel spreadsheet that includes the 405 nm values for each well at every timepoint. You are responsible for the data analysis of only your samples, not for the entire plate.

1. Average the triplicate wells for each condition you tested at every timepoint.
2. Plot the averaged values (x-axis) over time (y-axis) for each condition.
3. Use the t = 0 and t = 30 to calculate the activity of FKBP12 condition tested.
   • TEST = Condition 2 - 5, and BLANK = Condition 1.
   • Be sure to use the appropriate value for minutes given the t values.
   • The calculations are completed for each condition separately.
4. Post the units / mg protein value calculated for each condition to Class data page.
   • In addition, email your spreadsheet (with the plot and calculations) to the teaching faculty.

Reagents

• FKBP12 (1 mg/mL), Abcam
• chymotrypsin, Sigma
• suc-AAFP-pNA, Sigma
• rapamycin, Sigma
• ligands, Chembridge

Navigation links

Next day: Confirm ligand binding using differential scanning fluorimetry assay