Screening Compounds of Interest against USP5 Zf-UBD by SPR #2

I used a surface plasmon resonance assay to determine the binding affinities of hit compounds that were identified in 19F NMR spectroscopy screens against the zinc finger ubiquitin binding domain of USP5. To see how these assays work, check out some of my previous posts! You can see details of the SPR experiment on Zenodo. Read More …

Developing a Tryptophan Fluorescence Assay for Screening Ligands against USP5 Zf-UBD

For those following the USP5 project on my open notebook, you will recognize that assay development has been quite a challenge for screening ligands against USP5 zinc finger ubiquitin binding domain (Zf-UBD). In the past, I was unsuccessful in developing a differential scanning fluorimetry, and fluorescence polarization screening assay. I then tried 19F NMR spectroscopy screening, Read More …

Screening Compounds against USP5 Zf-UBD with a 19F NMR assay #2

I used a medium-throughput 19F NMR assay to screen my second batch of compounds against USP5 zinc finger ubiquitin binding domain (Zf-UBD). To recap, the 19F NMR assay detects ligand binding by measuring perturbations in the resonance of a fluorinated tryptophan at the binding pocket. I selected the compounds for my second screen from SGC’s in Read More …

Screening compounds of interest against USP5 Zf-UBD with a surface plasmon resonance assay

It’s been a busy couple of weeks in and out of the lab for me which is why it’s been some time since my last post. I previously identified small molecule compounds of interest using a 19F NMR spectroscopy assay. To quantify and validate these small molecule compounds, I used a surface plasmon resonance (SPR) Read More …

Compounds of interest identified by screening a focused library against USP5 Zf-UBD with a 19F NMR assay

In my last post, I developed a low-throughput 19F NMR screening assay against USP5 Zf-UBD, where ligand binding of a ubiquitin peptide was detected by perturbations in the resonance of a fluorinated tryptophan at the binding pocket. I recently screened the first batch of compounds that Ivan Franzoni and Renato Freitas, post-docs at the University Read More …

USP5 Zf-UBD Differential Scanning Fluorimetry Assay Development #3

In my last post, I determined that fluorescence polarization with different lengths of ubiquitin peptides was not a viable assay to screen compounds. I wanted to see if the ubiquitin peptides of different lengths resulted in a thermal shift of the USP5 Zf-UBD in a differential scanning fluorimetry assay. Experimental details are on Zenodo. As Read More …

USP5 Zf-UBD Fluorescence Polarization Displacement Assay #3

In previous experiments, a FITC-RLRGG ubiquitin peptide had a Kd of ~55 µM for USP5 Zf-UBD. Different lengths of ubiquitin peptides were designed to see if increasing the peptide length resulted in a gain in affinity for the protein domain in the FP assay: LRLRGG, RAHGLRLRGG, RAHGRAKHGLRLRGG. The 6-mer peptide, LRLRGG, is the native C-terminal Read More …

Crystallization of USP5 Zf-UBD #2

In my last post, I analyzed a structure of USP5 zinc-finger ubiquitin binding domain with a cysteine-glutathione disulphide bond. These crystals were not suitable for ligand soaking due to the formation of a disulphide bond at Cys195 with a glutathione additive that occupies the ubiquitin binding pocket in the adjacent molecule in the crystal lattice. Read More …