A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase.

Kaschner M, Schillinger O, Fettweiss T, Nutschel C, Krause F, Fulton A, Strodel B, Stadler A, Jaeger KE, Krauss U, Sci Rep 7:42592 (2017) Europe PMC

SASDDH7 – Citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA in the presence of citrate

citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA
MWI(0) 80 kDa
MWexpected 77 kDa
log I(s) 8.01×101 8.01×100 8.01×10-1 8.01×10-2
citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA small angle scattering data  s, nm-1
ln I(s)
citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA Guinier plot ln 8.02×101 Rg: 3.3 nm 0 (3.3 nm)-2 s2
(sRg)2I(s)/I(0)
citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA Kratky plot 1.104 0 √3 sRg
p(r)
citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA pair distance distribution function Rg: 3.4 nm 0 Dmax: 11.7 nm

Data validation

Fits and models

log I(s)
 s, nm-1
citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA GROMACS model
log I(s)
 s, nm-1
citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA DAMMIF model
citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA DAMMIF model
Synchrotron SAXS data from solutions of the citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA in 10 mM glycine buffer, 10 mM NaCl, 1 mM sodium citrate, pH 10 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.8 m and at a wavelength of λ = 0.09919 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 5.01 mg/ml was measured at 20°C. 10 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.

The protein is a fusion of: i) CitAP - the PAS domain from Sensor histidine kinase, CitA, from Klebsiella pneumoniae; UniProt: P52687 (Amino acid range 45-177); ii) BsLA - the Lipase EstA from Bacillus subtilis 168; UniProt: I6V559 (Amino acid range 32-212) and; iii) A linker connecting CitAP with EstA - derived from the blue light photoreceptor, YtvA, from Bacillus subtilis 168; UniProt: O34627 (Amino acid range 132-147).

citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA (CitAP-BsLA)
Mol. type   Protein
Olig. state   Dimer
Mon. MW   38.5 kDa
Sequence   FASTA