X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin

Campos-Escamilla C, Siliqi D, Gonzalez-Ramirez L, Lopez-Sanchez C, Gavira J, Moreno A, International Journal of Molecular Sciences 22(24):13392 (2021) DOI

SASDMS7 – HOLO-serotransferrin at pH 8.0 in the presence of iron

MWexperimental 66 kDa
MWexpected 77 kDa
VPorod 106 nm3
log I(s) 2.42×102 2.42×101 2.42×100 2.42×10-1
Serotransferrin small angle scattering data  s, nm-1
ln I(s)
Serotransferrin Guinier plot ln 2.42×102 Rg: 3.2 nm 0 (3.2 nm)-2 s2
Serotransferrin Kratky plot 1.104 0 3 sRg
Serotransferrin pair distance distribution function Rg: 3.3 nm 0 Dmax: 13.1 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Serotransferrin GASBOR model

log I(s)
 s, nm-1
Serotransferrin PDB (PROTEIN DATA BANK) model

log I(s)
 s, nm-1
Serotransferrin SREFLEX model

SAXS experiments for transferrin were carried out at BM29 beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The wavelength of incident X-rays was 0.1 nm and the Pilatus3 2M detector was placed 2.87 m from the sample leading to a range of the scattering vector from 0.025 to 6 nm-1. To avoid radiation damage, samples with a volume of 30-40 ┬ÁL were measured using a robotic sample handler in flow-through mode, collecting over 10 frames lasting 1 s for each sample. Frames were automatically checked for radiation damage and those not displaying any radiation damage were then averaged. Before and after each sample, buffer scattering was collected and subtracted from sample scattering. The buffer consisted of 15 mM HEPES, 20 mM NaHCO3, and 50 mM NaCl, tested at pH 8.0, 7.0, and 5.5. To assess concentration effects, a dilution series consisting of 2 concentrations (2.5 and 5 mg ml-1), for both Tf-apo and Tf-holo samples, was measured. Since the scattering curves for Tf did not display any concentration dependence at the highest concentrations, this concentration (5 mg ml-1) was used in our analysis. Merging, subtracting and subsequent analysis was performed using PRIMUS and Scatter software. A MX model (PDB ID: 2HAV) was compared with the SAXS data, including a modelling by GASBOR (top). For holo-Tf the MX model (middle) does not fit the SAXS data and it's dependent on pH changes. This indicates the change of the conformation in the presence of iron. The MX model was further refined by SREFLEX (bottom).

Serotransferrin (Tf)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   77.1 kDa
UniProt   P02787 (1-698)
Sequence   FASTA