In my continued quest to develop biophysical assays to screen compounds against USP5 Zf-UBD, I have been working on optimizing fluorescence polarization (FP) assay conditions for a peptide displacement assay. The aim was to optimize buffer conditions to decrease the amount of protein required for the assay, and to determine the affinity of a ubiquitin peptide, RLRGG to the protein domain. Experimental details can be found on Zenodo.
I started with using a buffer determined in a previous post and tested USP5 Zf-UBD concentrations with a fluorescently labeled ubiquitin peptide in various conditions. By optimizing the buffer, I was able to determine the Kdisp of the ubiquitin peptide to be ~55 µM; however, the assay with the peptide I used is not ideal for screening compounds due to the low dynamic range of polarization as indicated by a Z-factor of 0.39. The Z-factor is a measure of statistical effect, and is used to determine if high throughput screening is viable for an assay; typically a Z-factor greater than 0.5 indicates the signal to noise ratio in the assay is large enough to screen compounds. In the future, I hope to test this assay with ubiquitin peptides of various lengths. On one hand, I want to increase the peptide length to increase its affinity for USP5 and the polarization signal, but on the other hand, if the peptide is too large, the molecular weight ratio of the free peptide versus the USP5-peptide complex will decrease and so will polarization. Hopefully, I can find a good compromise.
Figure 1. a) RLRGG peptide titration in 50 mM bis-tris propane, 50 mM NaCl, 1 mM DTT, 0.01% Triton X-100 with 20 µM USP5 Zf-UBD and 40 nM FITC-RLRGG; b) Z-factor determination of USP5 Zf-UBD with FITC-RLRGG