Small-angle X-ray scattering analysis of circularly permuted carboxypeptidase G2 pro-enzymes

Brahm Yachnin.

SASDLN2 – Pro-CPG2-1-Disulfide (pro-enzyme design 1 disulfide variant of circular permutant Carboxypeptidase G2-CP-N89-K177A) with Methotrexate

Methotrexate
Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant
MWexperimental 96 kDa
MWexpected 97 kDa
VPorod 125 nm3
log I(s) 5.39×101 5.39×100 5.39×10-1 5.39×10-2
Methotrexate Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant small angle scattering data  s, nm-1
ln I(s)
Methotrexate Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant Guinier plot ln 5.39×101 Rg: 3.5 nm 0 (3.5 nm)-2 s2
(sRg)2I(s)/I(0)
Methotrexate Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant Kratky plot 1.104 0 3 sRg
p(r)
Methotrexate Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant pair distance distribution function Rg: 3.5 nm 0 Dmax: 12.3 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Methotrexate Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant ROSETTA model
Methotrexate Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant ROSETTA model

Synchrotron SAXS data from solutions of Pro-CPG2-1-Disulfide (pro-enzyme design 1 disulfide variant of circular permutant Carboxypeptidase G2-CP-N89-K177A) with Methotrexate in 50 mM Tris, 100 mM NaCl, pH 7.4 were collected on the 12.3.1 (SIBYLS) beam line at the Advanced Light Source (ALS) storage ring (Berkeley, CA, USA) using a Pilatus3 X 2M detector (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 2 and 10 mg/ml were measured at 10°C. 32 successive 0.300 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. The low angle data collected at lower concentration were merged with the highest concentration high angle data to yield the final composite scattering curve.

Merged SAXS scattering curve of the Pro-CPG2-1 disulfide variant pro-enzyme design of CP-N89 circular permutant of carboxypeptidase G2 with the K177A, A90C, and M424C (wild-type residue numbering) mutations in the presence of methotrexate. (Methotrexate is expected to have been converted to its cleaved form prior to data collection.) X-ray wavelength: UNKNOWN.

Methotrexate (MTX)
Mol. type   Other
Olig. state   Dimer
Mon. MW   0.5 kDa
Chemical formula
 
Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant (Pro-CPG2-1-Disulfide)
Mol. type   Protein
Organism   Pseudomonas sp. (strain RS-16)
Olig. state   Dimer
Mon. MW   48.0 kDa
 
UniProt   P06621 (89-415)
Sequence   FASTA
 
PDB code   1CG2
 
PDB code   1CG2