Structure-based screening of binding affinities via small-angle X-ray scattering

Chen P, Masiewicz P, Perez K, Hennig J, (2019) DOI

SASDFE8 – Histidine-binding periplasmic protein (HisBP) in the presence of histidine - His-bound 5-fold excess

Histidine-binding periplasmic protein
MWexperimental 30 kDa
MWexpected 26 kDa
VPorod 33 nm3
log I(s) 1.28×102 1.28×101 1.28×100 1.28×10-1
Histidine-binding periplasmic protein small angle scattering data  s, nm-1
ln I(s)
Histidine-binding periplasmic protein Guinier plot ln 1.29×102 Rg: 1.8 nm 0 (1.8 nm)-2 s2
(sRg)2I(s)/I(0)
Histidine-binding periplasmic protein Kratky plot 1.104 0 3 sRg
p(r)
Histidine-binding periplasmic protein pair distance distribution function Rg: 1.8 nm 0 Dmax: 5.7 nm

Data validation


There are no models related to this curve.

Synchrotron SAXS data from solutions of HisBP with a 5-fold excess of histidine in 100 mM NaCl, 20 mM NaPO4, 0.5 mM TCEP, pH 7.4 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.8 m and at a wavelength of λ = 0.09919 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 4.20 mg/ml was measured at 20°C. 12 successive 2 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.

His-bound HisBP at a ligand:protein ratio of 5:1. Ligand condition is prepared by dissolving histidine in powder form (Sigma-Aldrich) directly into the SAXS buffer, followed by dilution to the target concentration with additional SAXS buffer.

Histidine-binding periplasmic protein (HisBP)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Monomer
Mon. MW   26.3 kDa
 
UniProt   P0AEU0 (23-260)
Sequence   FASTA