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

Brahm Yachnin.

SASDLM2 – Pro-CPG2-1-Disulfide (pro-enzyme design 1 disulfide variant of circular permutant Carboxypeptidase G2-CP-N89-K177A)

Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant
MWexperimental 96 kDa
MWexpected 96 kDa
VPorod 120 nm3
log I(s) 2.28×102 2.28×101 2.28×100 2.28×10-1
Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant small angle scattering data  s, nm-1
ln I(s)
Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant Guinier plot ln 2.29×102 Rg: 3.6 nm 0 (3.6 nm)-2 s2
(sRg)2I(s)/I(0)
Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant Kratky plot 1.104 0 3 sRg
p(r)
Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant pair distance distribution function Rg: 3.6 nm 0 Dmax: 12.6 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Pro-Carboxypeptidase G2 (circular permutant CP-N89) K177A Design 1 Disulfide Variant ROSETTA model
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) 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. X-ray wavelength: UNKNOWN.

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