Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units

Galagedera S, Dao T, Enos S, Chaudhuri A, Schmit J, CastaƱeda C, Proceedings of the National Academy of Sciences 120(42) (2023) DOI

SASDR57 – HOTag6 tetramerization domain followed by a (Gly-Ser)4 linker fused to human monoubiquitin

MWI(0) 52 kDa
MWexpected 53 kDa
VPorod 76 nm3
log I(s) 2.15×10-2 2.15×10-3 2.15×10-4 2.15×10-5
HOTag-(GS)4-Ubiquitin small angle scattering data  s, nm-1
ln I(s)
HOTag-(GS)4-Ubiquitin Guinier plot ln 2.16×10-2 Rg: 3.8 nm 0 (3.8 nm)-2 s2
HOTag-(GS)4-Ubiquitin Kratky plot 1.104 0 3 sRg
HOTag-(GS)4-Ubiquitin pair distance distribution function Rg: 3.9 nm 0 Dmax: 14.3 nm

Data validation

There are no models related to this curve.

Synchrotron SAXS data from solutions of HOTag6 tetramerization domain followed by a (Gly-Ser)4 linker fused to human monoubiquitin in 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH 6.8 were collected on the BioCAT 18ID beam line at the Advanced Photon Source (APS), Argonne National Laboratory storage ring (Lemont, IL, USA) using a Pilatus3 X 1M detector at a sample-detector distance of 3.7 m and at a wavelength of λ = 0.1033 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 300.00 μl sample at 4.5 mg/ml was injected at a 0.60 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 20°C. 52 successive 0.500 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

HOTag6 is a ~30-amino-acid tetramerization domain followed by a (Gly-Ser)4 linker followed by the sequence for human monoubiquitin (UniProt ID: P0CG47, https://www.uniprot.org/uniprot/P0CG47, amino acids 1-76) . This is a designed protein (no natural variant exists). The HOTag6 sequence is from the Shu group at the University of California, San Francisco (https://doi.org/10.1016/j.molcel.2017.12.008).

Mol. type   Protein
Organism   Homo sapiens
Olig. state   Tetramer
Mon. MW   13.3 kDa
Sequence   FASTA