SEC-MALS: USP5 and Ubiquitin

Hi all! Hope everyone is staying safe and practising social distancing during these difficult times. Working from home, I’ve decided to catch up on my open notebook.

In a previous post, I purified various full length USP5 constructs and determined the binding affinity of ubiquitin to these constructs. I wanted to determine the stoichiometry of ubiquitin (Ubq) binding to USP5, as USP5 has two ubiquitin-associated domains, in addition to its canonical zinc-finger ubiquitin binding domain (ZnF-UBD) and a cryptic ZnF-UBP domain (Figure 1)1.

Figure 1. Schematic of full-length USP5 (grey) bound to ubiquitin (blue) (PDB: 3IHP): canonical ZnF-UBD (purple), cryptic ZnF-UBP (light blue), ubiquitin-associated domain-1 (yellow), ubiquitin-associated domain-2 (orange)

I used size exclusion chromatography-multiple angle light scattering (SEC-MALS) to determine the molar mass of the full-length USP5 protein in the presence and absence of an excess of monoubiquitin. MALS measures the light scattered by a protein at a range of angles allowing for extrapolation to determine molecular weight using a refractive index (RI) detector. Please see experimental details on Zenodo

I looked at two full-length USP5 constructs: wildtype (WT) USP5, and an R221A mutant (this is a mutation at the canonical ZnF-UBD) in the presence and absence of ubiquitin (Figure 2 & 3). The molecular weight is summarized in Table 1.

Table 1. Summary of Peak Molar Mass Moment and Molecular Weight

Mp (g/mol) Peak Molecular Weight (kDa)
WT USP5 9.970 x 104 (±0.271%) 99.7
WT USP5 + Ubq 1.068 x 105 (±0.338%) 106.8
R221A USP5 1.064 x 105 (±0.321%) 106.4
R221A USP5 + Ubq 1.059 x 105 (±0.351%) 105.9

Figure 2. SEC-MALS of wildtype USP5 only (black) and wildtype USP5 + Ubq (blue)

Figure 3. SEC-MALS of R221A mutant USP5 only (black) and R221A USP5 + Ubq (blue)

WT USP5 is fairly monodispersed (Mw/Mn= 1.1) and has a molecular weight of approximately 99 kDa, similar to the expected molecular weight of the protein which is 95.3 k Da. WT USP5 + Ubq elutes at the same volume as WT USP5 (17.4 mL) and has a molecular weight of approximately 107 kDa. This corresponds to 1:1 binding of ubiquitin (MW= 8.6 kDa) to USP5.

R221A USP5 has an approximate molecular weight of 106 kDa, which is higher than the expected molecular weight of the protein (MW= 99.6 kDa). R221A + Ubq also has an approximate molecular weight of 106 kDa. The mutant R221A ± Ubq are not significantly different and without orthogonal experiments, it is difficult to interpret what the MALS data means. Interestingly, there was a shift in the elution volume of the R221A + Ubq compared to R221A, where the R221A + Ubq elutes approximately 0.2 mL before R221A USP5. Perhaps, there is a conformational shift of the R221A USP5 in the presence of ubiquitin. Large conformational remodeling of full-length USP5 was previously suggested1.

Next, we will use dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) to better understand ubiquitin binding to various USP5 constructs, including R221A. Stay tuned!


1Biochemistry 2012, 51, 6, 1188-1198

UniProt ID: P45974

Leave a Reply

Your email address will not be published. Required fields are marked *