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

SASDJ69 – Sensory rhodopsin II - transducer complex (NpSRII/NpHtrII) in detergent at 150 mM NaCl studied with SAXS

Sensory rhodopsin II from Natronbacterium pharaonis
Sensory rhodopsin II transducer from Natronomonas pharaonis
MWexperimental 310 kDa
MWexpected 169 kDa
log I(s) 1.13×10-1 1.13×10-2 1.13×10-3 1.13×10-4
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 1.13×10-1 Rg: 7.6 nm 0 (7.6 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: 10.2 nm 0 Dmax: 41 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 MEMPROT model
log I(s)
 s, nm-1
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis DAMMIF model
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis DAMFILT model
log I(s)
 s, nm-1
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis DAMMIF model
Sensory rhodopsin II from Natronbacterium pharaonis Sensory rhodopsin II transducer from Natronomonas pharaonis DAMFILT model
Synchrotron SAXS data from solutions of Sensory rhodopsin II - transducer complex (NpSRII/NpHtrII) in detergent at 150 mM NaCl studied with SAXS in 150 mM NaCl, 25 mM Na/Na-Pi, 1.0 mM EDTA, 0.05% DDM, pH 8 were collected on the BM29 beam line at the ESRF storage ring (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.9918 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 0.5 and 0.6 mg/ml were measured at 20°C. 16 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 low angle data collected at lower concentration were merged with the highest concentration high angle data to yield the final composite scattering curve.

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.

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