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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)

10% glycerol

ATP regeneration mix (1X)

4 mM ATP - must be pH to 7.0

16 mM creatine phosphate

0.32 mg/mL creatine kinase


Reaction

1X PD buffer

200 mM KCl

1X ATP regeneration mix

800 nM ClpX6

300 nM ClpP14

0.5 - 120 µM peptide

Measuring degradation

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

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.