Mandeep Mann

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 …

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 …

Based on previous 19F NMR and SPR assay experiments, 4 compounds were used to set up co-crystallization experiments (Table 1). I was able to get co-crystal structures of 3 of the 4 compounds! You can see full experimental details on Zenodo. Table 1. USP5 Zf-UBD ligands                   Read More …

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 …

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 …

I have had little success with DSF and FP assay development for USP5 Zf-UBD to identify small molecule ligands of this domain. I decided to try 19F NMR as a potential screening assay. NMR spectroscopy can be used to visualize changes in protein spectra upon addition of low complexity molecules as well as to quantify Read More …

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 …

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 …

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 …

In a previous post, I determined a crystallization condition that produced nice 3D crystals of USP5 Zf-UBD that diffracted well. Rachel Harding, a postdoc at the SGC collected data images for these crystals and solved the structure using computational techniques. You can see the solved crystal structure and a more detailed analysis of the structure Read More …