Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer.

Golovinas E, Rutkauskas D, Manakova E, Jankunec M, Silanskas A, Sasnauskas G, Zaremba M, Sci Rep 11(1):4518 (2021) Europe PMC

SASDH49 – Complex of Archaeoglobus fulgidus Argonaute monomeric mutant with dsDNA (5'p-ATTGTACGTACAAT)

Piwi protein AF_1318 delta (296-303) mutant
5'-phosphorilated 14-mer DNA oligoduplex
MWI(0) 55 kDa
MWexpected 59 kDa
VPorod 109 nm3
log I(s) 4.28×10-2 4.28×10-3 4.28×10-4 4.28×10-5
Piwi protein AF_1318 delta (296-303) mutant 5'-phosphorilated 14-mer DNA oligoduplex small angle scattering data  s, nm-1
ln I(s)
Piwi protein AF_1318 delta (296-303) mutant 5'-phosphorilated 14-mer DNA oligoduplex Guinier plot ln 4.28×10-2 Rg: 2.8 nm 0 (2.8 nm)-2 s2
(sRg)2I(s)/I(0)
Piwi protein AF_1318 delta (296-303) mutant 5'-phosphorilated 14-mer DNA oligoduplex Kratky plot 1.104 0 √3 sRg
p(r)
Piwi protein AF_1318 delta (296-303) mutant 5'-phosphorilated 14-mer DNA oligoduplex pair distance distribution function Rg: 2.9 nm 0 Dmax: 9.6 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Piwi protein AF_1318 delta (296-303) mutant 5'-phosphorilated 14-mer DNA oligoduplex PDB (PROTEIN DATA BANK) model
log I(s)
 s, nm-1
Piwi protein AF_1318 delta (296-303) mutant 5'-phosphorilated 14-mer DNA oligoduplex GASBOR model
Synchrotron SAXS data from solutions of mutant Argonaute bound to dsDNA (5'p-ATTGTACGTACAAT) in 20 mM TrisHCl, pH 7.5, 5 mM MgCl2, 150 mM NaCl and 2 mM DTT were collected on the EMBL P12 beam line at PETRA III (DESY, Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 4.00 mg/ml was measured at 20°C. 30 successive 0.195 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.

Monomeric AfAgo complexed with dsDNA shows moderate aggregation, therefore the first 39 low-angle points of the SAXS data were removed.

Piwi protein AF_1318 delta (296-303) mutant (AfAgo delta(296-303))
Mol. type   Protein
Organism   Archaeoglobus fulgidus
Olig. state   Monomer
Mon. MW   49.9 kDa
Sequence   FASTA
 
5'-phosphorilated 14-mer DNA oligoduplex (MZ1289)
Mol. type   DNA
Olig. state   Dimer
Mon. MW   4.3 kDa
Sequence   FASTA
 
PDB ID   2W42