Ferritin-based fusion protein shows octameric deadlock state of self-assembly

Sudarev V, Gette M, Bazhenov S, Tilinova O, Zinovev E, Manukhov I, Kuklin A, Ryzhykau Y, Vlasov A, Biochemical and Biophysical Research Communications 690:149276 (2024) DOI

SASDTV4 – 8×FerSUMO - Chimeric protein complex of ferritin from Helicobacter pylori fused to an N-terminal His6-SUMO protein tag

Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion)
MWexperimental 252 kDa
MWexpected 252 kDa
VPorod 618 nm3
log I(s) 1.77×10-1 1.77×10-2 1.77×10-3 1.77×10-4
Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion) small angle scattering data  s, nm-1
ln I(s)
Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion) Guinier plot ln 1.77×10-1 Rg: 5.4 nm 0 (5.4 nm)-2 s2
(sRg)2I(s)/I(0)
Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion) Kratky plot 1.104 0 √3 sRg
p(r)
Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion) pair distance distribution function Rg: 5.1 nm 0 Dmax: 16 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion) PHYRE2 model
log I(s)
 s, nm-1
Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion) ALPHAFOLD model
SAXS data from solutions of 8×FerSUMO in 25 mM Tris, 150 mM NaCl, pH 7.4 were collected using a Rigaku MicroMax 007-HF instrument at the Moscow Institute of Physics and Technology (MIPT; Dolgoprudny, Russian Federation) equipped with a multiwire gas-filled ASM DTR Triton 200 detector at a sample-detector distance of 2 m and at a wavelength of λ = 0.15406 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 5.00 mg/ml was measured at 20°C. One 9720 second frame was collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Chimeric protein complex of ferritin from Helicobacter pylori (UniProt ID: Q9ZLI1, corresponds to C-terminal region of obtained protein construct used for SAXS) with N-terminal His6-SUMO-tag. The SUMO-tag corresponds to UniProt ID Q12306, amino acids 3-98 with mutations R64T and R71E.

Bacterial non-heme ferritin (N19Q, I59V, N-terminal His-SUMO fusion) (Fe8×FerSUMO)
Mol. type   Protein
Organism   Helicobacter pylori (strain J99 / ATCC 700824)
Olig. state   Octamer
Mon. MW   31.5 kDa
 
UniProt   Q9ZLI1 (5-167)
Sequence   FASTA