Anti-Stokes fluorescence excitation reveals conformational mobility of the C-phycocyanin chromophores

Tsoraev G, Protasova E, Klimanova E, Ryzhykau Y, Kuklin A, Semenov Y, Ge B, Li W, Qin S, Friedrich T, Sluchanko N, Maksimov E, Structural Dynamics 9(5):054701 (2022) DOI

SASDPB4 – SAXS data for the C-phycocyanin in 8 M urea solution

C-phycocyanin alpha subunit
C-phycocyanin beta subunit
MWI(0) 65 kDa
MWexpected 36 kDa
log I(s) 4.05×10-1 4.05×10-2 4.05×10-3 4.05×10-4
C-phycocyanin alpha subunit C-phycocyanin beta subunit small angle scattering data  s, nm-1
ln I(s)
C-phycocyanin alpha subunit C-phycocyanin beta subunit Guinier plot ln 4.06×10-1 Rg: 6.6 nm 0 (6.6 nm)-2 s2
(sRg)2I(s)/I(0)
C-phycocyanin alpha subunit C-phycocyanin beta subunit Kratky plot 1.104 0 √3 sRg
p(r)
C-phycocyanin alpha subunit C-phycocyanin beta subunit pair distance distribution function Rg: 6.7 nm 0 Dmax: 23.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
C-phycocyanin alpha subunit C-phycocyanin beta subunit GASBOR model
SAXS data from solutions of SAXS data for the C-phycocyanin in 8 M urea solution in 8 M urea, 30 mM βME, pH 7 were measured using a Rigaku MicroMax 007-HF instrument at the Moscow Institute of Physics and Technology (MIPT; Russian Federation) equipped with a multiwire gas-filled ASM DTR Triton 200 detector at a sample-detector distance of 2 m and at a wavelength of λ = 0.154 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 20.00 mg/ml was measured at 20°C. One 18000 second frame was collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Spirulina platensis C-phycocyanin is composed of alpha and beta subunits. This complex forms ring-shaped trimers at 150 mM NaCl. When the protein is transferred into 8 M urea, it partially unfolds, which leads to disruption of intersubunit contacts. This results in partial dissociation of the trimers into monomers and possibly also into separate alpha and beta subunits. The maximum diameter is 23.5 nm, which is almost twice the diameter of the ring-like trimer (Dmax = 13.2 nm), but is in good agreement with the “unrolled” ring model. The ab initio GASBOR structure is derived from SAXS data that corresponds to an ensemble of at least monomers and trimers with high conformational flexibility. Therefore, this model can only demonstrate that proteins are partially unfolded and have an elongated shape.

C-phycocyanin alpha subunit (cpcA)
Mol. type   Protein
Organism   Arthrospira platensis
Olig. state   Monomer
Mon. MW   17.6 kDa
 
UniProt   P72509 (1-162)
Sequence   FASTA
 
C-phycocyanin beta subunit (cpcB)
Mol. type   Protein
Organism   Arthrospira platensis
Olig. state   Monomer
Mon. MW   18.1 kDa
 
UniProt   P72508 (1-172)
Sequence   FASTA