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