USP5

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 …

I wanted to determine whether USP5 Zf-UBD is a monomer or a dimer in solution as some known USP5 structures have shown that there is a cysteine surface residue that is not involved in Zn-coordination but forms disulfide bonds (2G43, 2G45, 3IHP). I used size exclusion chromatography with multiple angle light scattering (SEC-MALS) to determine Read More …

In a previous post, I outlined initial experiments to develop a differential scanning fluorimetry (DSF) assay for USP5 Zf-UBD where I measure the resistance of USP5 to thermal denaturation as small molecules bind and stabilize the protein. I previously could not observe any significant change in the melting temperature of USP5, even using a ubiquitin Read More …

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 Read More …

It’s important to have more than one assay in place to validate experimental results. In addition to the fluorescence polarization assay, I am also trying to develop a differential scanning fluorimetry (DSF) assay. DSF measures the interaction of small molecules to the protein as the protein progressively unfolds. The melting temperature of USP5 Zf-UBD is Read More …

There have been some exciting developments in the lab since my last post…I’ve got apo crystals of USP5 Zf-UBD! Experimental details can be found on Zenodo. The apo structure, which has no ligands bound in the receptor pocket has already been solved. The purified protein sample I have been working with for USP5 Zf-UBD is Read More …

USP5 is one of many USP proteins that contains a zinc finger ubiquitin binding domain (Zf-UBD). My goal is to develop small molecules that inhibit USP5 selectively. So, it’s important to make sure that the pocket where the compound will bind is sufficiently and structurally different from other USPs. I did a multiple sequence alignment Read More …

For the past few days, I have been working on optimizing a fluorescence polarization (FP) displacement assay for USP5 Zf-UBD. This assay will be used to test if small molecules can displace a fluorescence-labelled ubiquitin peptide from USP5. I started with screening different assay conditions with a small set of buffers. The method and data Read More …

Before I can begin characterizing the USP5 protein, I first needed to make some protein sample to work with. I expressed and purified the zinc-finger domain protein in bacteria and ended up with a high yield of a sample which is very pure. All of my methods and data from this experiment can be found Read More …

Ubiquitination can mark proteins for degradation, alter their cellular location, affect their activity, and promote or prevent protein interactions. Ubiquitin specific proteases (USPs) are the largest family of deubiquitinase enzymes. USP5 disassembles unanchored ubiquitin chains. This prevents the accumulation of ubiquitin chains which would otherwise compete against ubiquitinated proteins for degradation as well as replenishes the Read More …