B. pertussis adenylate cyclase toxin-derived constructs

Carlos Espinosa-Vinals.

SASDPP3 – RTX719. Double-acylated truncated variant of B. pertussis adenylate cyclase toxin (CyaA)

Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294
MWexperimental 86 kDa
MWexpected 78 kDa
VPorod 67 nm3
log I(s) 4.34×10-2 4.34×10-3 4.34×10-4 4.34×10-5
Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 small angle scattering data  s, nm-1
ln I(s)
Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 Guinier plot ln 4.34×10-2 Rg: 3.4 nm 0 (3.4 nm)-2 s2
(sRg)2I(s)/I(0)
Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 Kratky plot 1.104 0 √3 sRg
p(r)
Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 pair distance distribution function Rg: 3.8 nm 0 Dmax: 14.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
RTX719. Double-acylated truncated variant of B. pertussis adenylate cyclase toxin (CyaA) Rg histogram Rg, nm
Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 EOM/RANCH model
Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 EOM/RANCH model
log I(s)
 s, nm-1
Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 ALPHAFOLD model
The SAXS measurements were done in a SAXSpoint 2.0 (Anton Paar, Graz, Austria) instrument, equipped with MetalJet C2 X-ray source (Excillum, Stockholm, Sweden) and Eiger R 1M (Dectris, Switzerland) detector. For the chromatographic separation of the oligomers and the monomeric species, an Akta GO (GE Healthcare) FPLC system with Superdex 200 Increase 10/300 column (GE Healthcare) was utilized. The chromatography was controlled automatically. The column was thoroughly equilibrated with the running buffer and the SAXS signal for buffer subtraction was also collected (2 hours) simultaneously. After loading the sample on the column, absorption of UV at 280nm was monitored (2 mm cell). Once the absorbance increased above 15 mAU, the flow rate in the system was reduced to 0.01ml/min and the sample was measured in a flow-through quartz capillary (2mm diameter), which enables in-situ UV-Vis spectroscopy using Cary 60 spectrometer (Agillent). SAXS and in-situ UV-Vis were continuously measured. The reduced scattering data was corrected to the fluctuation of primary beam, which was monitored using semi-transparent beamstop.

OVERSUBTRACTED.

Double-acylated variant of a genetic fusion comprising the C-terminal folding scaffold of RTX block V (residues 1562–1681 of CyaA) and the CyaA residues 719–1294 (RTX719)
Mol. type   Protein
Organism   Bordetella pertussis
Olig. state   Monomer
Mon. MW   78.0 kDa
Sequence   FASTA