Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Ryzhykau Y, Vlasov A, Orekhov P, Rulev M, Rogachev A, Vlasova A, Kazantsev A, Verteletskiy D, Skoi V, Brennich M, Pernot P, Murugova T, Gordeliy V, Kuklin A, Acta Crystallographica Section D Structural Biology 77(11) (2021) DOI

SASDJA9 – Sensory rhodopsin II - transducer complex (NpSRII/NpHtrII) in detergent at 4000 mM NaCl studied with SANS

Sensory rhodopsin II from Natronbacterium pharaonis
Sensory rhodopsin II transducer from Natronomonas pharaonis
MWexperimental 225 kDa
MWexpected 169 kDa
log I(s) 1.99×100 1.99×10-1 1.99×10-2 1.99×10-3
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis small angle scattering data  s, nm-1
ln I(s)
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis Guinier plot ln 1.99×100 Rg: 10.4 nm 0 (10.4 nm)-2 s2
(sRg)2I(s)/I(0)
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis Kratky plot 1.104 0 3 sRg
p(r)
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis pair distance distribution function Rg: 9.4 nm 0 Dmax: 39 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis GASBOR model

log I(s)
 s, nm-1
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis GASBOR model

SANS data from solutions of Sensory rhodopsin II - transducer complex (NpSRII/NpHtrII) in detergent at 4000 mM NaCl studied with SANS in 4000 mM NaCl, 100 mM Na/Na-Pi, 1.0 mM EDTA, 0.05% DDM (D2O buffer), pH 8 were collected using a He3-fulfilled, 8 independent wires detector at a sample-detector distance of 13.0 m and at a wavelength of λ = 0.147 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 0.40 mg/ml was measured at 20°C. Four successive 1800 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 of study is a non-fused complex of Sensory rhodopsin II (NpSRII, UniProt ID P42196) with its cognate transducer (NpHtrII, UniProt ID P42259) from Nartonomonas pharaonis. NpSRII/NpHtrII requires dimerization for signal transduction and forms a trimer of dimers in the N. pharaonis membrane. The dependence of the oligomeric state of this complex on conditions is the subject of research. The NpSRII/NpHtrII was co-expressed in E. coli and solubilized in n-Dodecyl β-D-maltoside; therefore, resulting molecular weight may differ from the expected due to the presence of a detergent belt surrounding the tramembrane domain of the complex. SANS measurements were performed on a YuMO (IBR-2) instrument with a two-detector system (sample-detector distances of 4.5 m and 12.97 m). The wavelengths used are from 0.5 to 8 Å, the contributions of which are separated using time-of-flight technology.

Sensory rhodopsin II from Natronbacterium pharaonis (NpSRII)
Mol. type   Protein
Organism   Natronomonas pharaonis
Olig. state   Dimer
Mon. MW   26.7 kDa
 
UniProt   P42196 (1-239)
Sequence   FASTA
 
Sensory rhodopsin II transducer from Natronomonas pharaonis (NpHtrII)
Mol. type   Protein
Organism   Natronomonas pharaonis
Olig. state   Dimer
Mon. MW   57.8 kDa
 
UniProt   P42259 (3-534)
Sequence   FASTA