Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations

Liu G, Ekmen E, Jalalypour F, Mertens H, Jeffries C, Svergun D, Atilgan A, Atilgan C, Sayers Z, The Journal of Chemical Physics 158(8) (2023) DOI

SASDWL7 – D52A mutant of ferric binding protein (FbpA) in apo (no Fe) form in high ionic strength (HIS) buffer (PBS)

Iron-utilization periplasmic protein
MWexperimental 32 kDa
MWexpected 34 kDa
VPorod 40 nm3
log I(s) 1.61×104 1.61×103 1.61×102 1.61×101
Iron-utilization periplasmic protein small angle scattering data  s, nm-1
ln I(s)
Iron-utilization periplasmic protein Guinier plot ln 1.61×104 Rg: 2.1 nm 0 (2.1 nm)-2 s2
(sRg)2I(s)/I(0)
Iron-utilization periplasmic protein Kratky plot 1.104 0 √3 sRg
p(r)
Iron-utilization periplasmic protein pair distance distribution function Rg: 2.0 nm 0 Dmax: 6.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Iron-utilization periplasmic protein DAMMIN model
Synchrotron SAXS data from solutions of D52A mutant of ferric binding protein (FbpA) in apo (no Fe) form in high ionic strength (HIS) buffer (PBS) in 10 mM Na2HPO4 . 7H2O 1.8 mM KH2PO4 137 mM NaCl 2.7 mM KCl 5% v/v Glycerol, pH 7.4 were collected on the EMBL P12 beam line at the PETRA III storage ring (DESY; Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.123987 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 75.00 μl sample at 12.9 mg/ml was injected at a 0.60 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 10°C. 2400 successive 0.995 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.

Iron-utilization periplasmic protein (FBPA_HAEIN)
Mol. type   Protein
Organism   Haemophilus influenzae
Olig. state   Monomer
Mon. MW   33.8 kDa
 
UniProt   P35755 (23-332)
Sequence   FASTA