Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA.

Miele AE, Cervoni L, Le Roy A, Cutone A, Musci G, Ebel C, Bonaccorsi di Patti MC, Eur Biophys J (2021) Europe PMC

SASDJ76 – Fep1 wild type with [2Fe2S] cluster reconstituted

GATA-type iron responsive transcription factor Fep1 reconstituted
MWexperimental 30 kDa
MWexpected 22 kDa
VPorod 53 nm3
log I(s) 6.73×100 6.73×10-1 6.73×10-2 6.73×10-3
GATA-type iron responsive transcription factor Fep1 reconstituted small angle scattering data  s, nm-1
ln I(s)
GATA-type iron responsive transcription factor Fep1 reconstituted Guinier plot ln 6.73×100 Rg: 3.8 nm 0 (3.8 nm)-2 s2
(sRg)2I(s)/I(0)
GATA-type iron responsive transcription factor Fep1 reconstituted Kratky plot 1.104 0 3 sRg
p(r)
GATA-type iron responsive transcription factor Fep1 reconstituted pair distance distribution function Rg: 4.3 nm 0 Dmax: 16.5 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Fep1 wild type with [2Fe2S] cluster reconstituted Rg histogram Rg, nm
GATA-type iron responsive transcription factor Fep1 reconstituted EOM/RANCH model
GATA-type iron responsive transcription factor Fep1 reconstituted EOM/RANCH model
GATA-type iron responsive transcription factor Fep1 reconstituted EOM/RANCH model
GATA-type iron responsive transcription factor Fep1 reconstituted EOM/RANCH model
GATA-type iron responsive transcription factor Fep1 reconstituted EOM/RANCH model
GATA-type iron responsive transcription factor Fep1 reconstituted EOM/RANCH model

Synchrotron SAXS data from solutions of Fep1 wild type with [2Fe2S] cluster reconstituted in 50 mM MOPS, 50 mM NaCl, pH 7 were collected on the BM29 beam line at the ESRF storage ring (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.0999 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 40.00 μl sample at 3.4 mg/ml was injected at a 0.40 ml/min flow rate onto a GE Superdex 75 Increase 5/150 column at 20°C. 900 successive 1 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.

Purified Fep1 was reconstituted with 2 stoichiometric excess of (FeCl3 and Na2S) in 50 mM MOPS, 50 mM NaCl, 1 mM TCEP pH 7.0, then the excess of Fe was eliminated by passing the solution into a PD10 column. Then the purified reconstituted protein was concentrated and the concentration was measured to be 3.43 mg/ml. Two successive HPLC run were performed on two injections by 40 microL each and the frames from the 2 main elution peaks were merged and analysed with Chromixs. The I(q) vs q were solvent subtracted by using the first 20 frames of the HPLC run, after being averaged. The same SEC column as for Fep1 wild type was used. Data were collected at BM29 of ESRF (Grenoble, France)

GATA-type iron responsive transcription factor Fep1 reconstituted (Fep1-rec)
Mol. type   Protein
Organism   Komagataella pastoris
Olig. state   Monomer
Mon. MW   22.1 kDa
 
UniProt   A4VAR6 (1-208)
Sequence   FASTA