Heme-substituted protein assembly bridged by synthetic porphyrin: achieving controlled configuration while maintaining rotational freedom

Inaba H, Shisaka Y, Ariyasu S, Sakakibara E, Ueda G, Aiba Y, Shimizu N, Sugimoto H, Shoji O, RSC Advances 14(13):8829-8836 (2024) DOI

SASDTK5 – Heme acquisition system protein A (39.9 kDa dimer from Pseudomonas protegens PF-5)

Heme acquisition protein HasAp
MWI(0) 39 kDa
MWexpected 38 kDa
VPorod 54 nm3
log I(s) 2.76×10-2 2.76×10-3 2.76×10-4 2.76×10-5
Heme acquisition protein HasAp small angle scattering data  s, nm-1
ln I(s)
Heme acquisition protein HasAp Guinier plot ln 2.76×10-2 Rg: 2.8 nm 0 (2.8 nm)-2 s2
(sRg)2I(s)/I(0)
Heme acquisition protein HasAp Kratky plot 1.104 0 √3 sRg
p(r)
Heme acquisition protein HasAp pair distance distribution function Rg: 2.8 nm 0 Dmax: 9.1 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Heme acquisition protein HasAp DAMMIN model
log I(s)
 s, nm-1
Heme acquisition protein HasAp OTHER model
log I(s)
 s, nm-1
Heme acquisition protein HasAp OTHER model
log I(s)
 s, nm-1
Heme acquisition protein HasAp OTHER model
log I(s)
 s, nm-1
Heme acquisition protein HasAp OTHER model
log I(s)
 s, nm-1
Heme acquisition protein HasAp OTHER model
log I(s)
 s, nm-1
Heme acquisition protein HasAp OTHER model
Synchrotron SAXS data from solutions of heme acquisition system protein A in 50 mM CHES, 5 % glycerol, pH 9.5 were collected on the BL-10C beam line at the Photon Factory (PF), High Energy Accelerator Research Organization (KEK; Tsukuba, Japan) using a Pilatus3 2M detector at a sample-detector distance of 2.1 m and at a wavelength of λ = 0.1 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 440.00 μl sample at 8 mg/ml was injected at a 0.05 ml/min flow rate onto a Cytiva Superdex 200 Increase 10/300 column at 20°C. 408 successive 20 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.

HasApf5 incorporating iron(III)-tetraphenylporphyrin (TPP) with the phenyl ligand attached to the phenyl group of Fe-TPP via a planar amide bond (Fe-TPP-phen) formed via a dimer of Ni2+ ions. The molecular weight of the dimer containing Fe-TPP-phen was estimated to be 39.9 kDa. This SAXS profile was compared with six atomic model structures (delta-cis.pdb, delta-trans1.pdb, delta-trans2.pdb, lambda-cis.pdb, lambda-trans1.pdb, lambda-trans2.pdb) that were created based on crystal structures. The methods to create these model structures are described in detail in the Supplementary Information of the article. Since lambda-trans1.pdb was shown to be the closest to the experimental results on the basis of the CRYSOL calculations, the remaining five models were superimposed on lambda-trans1.pdb using the software BIOVIA Discovery Studio.

Heme acquisition protein HasAp (HasApf5)
Mol. type   Protein
Organism   Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
Olig. state   Dimer
Mon. MW   19.0 kDa
 
UniProt   Q4K5N8 (1-183)
Sequence   FASTA