Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility

Orr C, Fisher H, Yu X, Chan C, Gao Y, Duriez P, Booth S, Elliott I, Inzhelevskaya T, Mockridge I, Penfold C, Wagner A, Glennie M, White A, Essex J, Pearson A, Cragg M, Tews I, Science Immunology 7(73) (2022) DOI

SASDLH4 – Human immunoglobulin gamma 2 (IgG2) ChiLob 7/4 F(ab)2 - Heavy chain C225S mutant

ChiLob 7/4 IgG2 F(ab)2 C225S mutant
MWexperimental 105 kDa
MWexpected 99 kDa
VPorod 131 nm3
log I(s) 3.43×101 3.43×100 3.43×10-1 3.43×10-2
ChiLob 7/4 IgG2 F(ab)2 C225S mutant small angle scattering data  s, nm-1
ln I(s)
ChiLob 7/4 IgG2 F(ab)2 C225S mutant Guinier plot ln 3.43×101 Rg: 4.0 nm 0 (4.0 nm)-2 s2
(sRg)2I(s)/I(0)
ChiLob 7/4 IgG2 F(ab)2 C225S mutant Kratky plot 1.104 0 √3 sRg
p(r)
ChiLob 7/4 IgG2 F(ab)2 C225S mutant pair distance distribution function Rg: 4.2 nm 0 Dmax: 14.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Human immunoglobulin gamma 2 (IgG2) ChiLob 7/4 F(ab)2 - Heavy chain C225S mutant Rg histogram Rg, nm
ChiLob 7/4 IgG2 F(ab)2 C225S mutant MOLECULAR DYNAMICS FRAME model
ChiLob 7/4 IgG2 F(ab)2 C225S mutant MOLECULAR DYNAMICS FRAME model
ChiLob 7/4 IgG2 F(ab)2 C225S mutant MOLECULAR DYNAMICS FRAME model
ChiLob 7/4 IgG2 F(ab)2 C225S mutant MOLECULAR DYNAMICS FRAME model
log I(s)
 s, nm-1
ChiLob 7/4 IgG2 F(ab)2 C225S mutant MODELLER model
log I(s)
 s, nm-1
ChiLob 7/4 IgG2 F(ab)2 C225S mutant SREFLEX model
Synchrotron SAXS data from solutions of Human immunoglobulin gamma 2 (IgG2) ChiLob 7/4 F(ab)2 - Heavy chain C225S mutant in 50 mM HEPES, 150 mM KCl, pH 7.5 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 = 0.099 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 45.00 μl sample at 26.6 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superdex 200 10/300 column at 25°C. 2700 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.

ChiLob 7/4 IgG2 F(ab)2 C225S mutant (C225S)
Mol. type   Protein
Olig. state   Monomer
Mon. MW   98.7 kDa
Sequence   FASTA
 
PDB ID   6TKC
 
PDB ID   6TKC
 
PDB ID   6TKC
 
PDB ID   6TKC
 
PDB ID   6TKC
 
PDB ID   6TKC