Sauer:In vitro peptide degradation by ClpXP

SAUER LAB

Home        Protocols        Lab Members        Materials        Equipment        Links        Internal       

Degradation of peptides containing Abz and Y(NO₂) by ClpXP

Peptides need to be HPLC purified and buffered prior to use. (Trace TFA can remain after HPLC purification, significantly affecting the pH.)

Peptide concentration can be determined by UV. ε₃₈₁= 2200 cm^-1

PD buffer (1X)

25 mM HEPES-KOH, pH 7.6

5 mM MgCl₂

0.032% NP-40 (Nonidet P40 substitute)

• Many people leave out the detergent and see similar activity, but it helps prevent protein sticking to tubes *

10% glycerol

• Make 4X stock and store at -20 ˚C.

ATP regeneration mix (1X)

4 mM ATP - must be pH to 7.0

16 mM creatine phosphate

0.32 mg/mL creatine kinase

• Make 10X stock and store at -20 ˚C.

Reaction

1X PD buffer

200 mM KCl

1X ATP regeneration mix

800 nM ClpX₆

300 nM ClpP₁₄

0.5 - 120 µM peptide

• Internal quenching throws off the degradation results above ~120 μM
• This results in double-capped ClpP. For single capped, use 800 nm ClpP and 300 nm ClpX instead.
• For peptide degradation followed by fluorescence, use 60 µL reaction, 0.3 mm cuvette.
• If you are using SspB, include it at a concentration that is equal to that of the peptide you are using.
• Be sure to equalize the salts that may come in with your proteins/peptides (SspB, etc.) across reactions because ClpX is very salt sensitive.
• Make a mix of everything except peptide and SspB such that you will add an equal amount of the master mix to each reaction. I usually make my Substrate+SspB equal to 20 µL and add 40 µL of the mix to each reaction.--
• Keep everything on ice until you are ready to do the reaction.

Measuring degradation

• Get instructions on how to use the fluorimeter.
• excitation wavelength = 320 nm, emission wavelength = 420 nm
• water bath at 30 ˚C

Pre-warm cuvette in cuvette holder for at least 5 min prior to each reaction.

Pre-warm peptide (and SspB, if using) in water bath for 2 min.

Pre-warm ClpX mix for reaction in a tube in the water bath or in cuvette.

Add substrate to ClpX in cuvette and mix well, trying not to introduce any bubbles.

Start measuring fluorescence and measure for ~3 min to get initial rate (beware of bubbles of equilibration artifacts).

Slit width

• Set all four the same, it's easier that way.
• I recommend ~ 3.5 half turns of the screw.

Correlating Rates with Concentration

You will need an end point for the degradation of each peptide to obtain the relationship between fluorescent counts and concentration. This can be obtained by letting the degradation go to completion with ClpXP, or by designing your fluorophore-quencher region such that either trypsin or chymotrypsin will cleave the peptide.

If you use either trypsin or chymotrypsin you will need to wash the cuvette well. This can be done by washing the cuvette, letting it sit with 1M HCL, and then washing it again to remove trace HCl.