Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs.

Josts I, Nitsche J, Maric S, Mertens HD, Moulin M, Haertlein M, Prevost S, Svergun DI, Busch S, Forsyth VT, Tidow H, Structure (2018) PubMed

SASDDB5 – MsbA in stealth nanodisc (SANS, 100% D2O)

Lipid A export ATP-binding/permease protein MsbA
Membrane scaffold protein 1D1 (deuterated, 75%)
1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl)
MWI(0) 145 kDa
MWexpected 158 kDa
VPorod 189 nm3
log I(s) 8.90×10-1 8.90×10-2 8.90×10-3 8.90×10-4
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) small angle scattering data  s, nm-1
ln I(s)
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Guinier plot ln 8.90×10-1 Rg: 4 nm 0 (4 nm)-2 s2
(sRg)2I(s)/I(0)
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Kratky plot 1.104 0 3 sRg
p(r)
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) pair distance distribution function Rg: 4.0 nm 0 Dmax: 13 nm

Experimental data validation


Fits and models


log I(s)
 s, nm-1
Lipid A export ATP-binding/permease protein MsbA Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) SASREF CV model
SANS data from solutions of MsbA in stealth nanodisc (SANS, 100% D2O) in 30 mM Tris, 150 mM NaCl, pH 7.5 were collected on the D11 camera at the ILL storage ring (Grenoble, France) using a 3-He gas detector CERCA detector at a wavelength of λ = 0.46 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 1 and 6 mg/ml were measured at 10°C. Two successive 4200 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.

MsbA ABC transporter protein encapsulated in a partially deuterated "stealth-nanodisc": (sND). Disc components (MSP1D1 scaffold protein - 75% deuterated, and POPC lipid - heads/tails 78% and 92% deuterated) are effectively matched out at 100% D2O. Thus the expected molecular weight should correspond to that of the MsbA protein (~132 kDa). Data measured in two configurations: 1.5 m / 1.5 m and 8 m / 8 m (collimation length / sample-detector distances) with a fixed neutron wavelength (λ) of 5 Å. Data is merged from these two configurations.

Lipid A export ATP-binding/permease protein MsbA (MsbA)
Mol. type   Protein
Organism   Escherichia coli (strain K12)
Olig. state   Dimer
Mon. MW   66.3 kDa
 
UniProt   P60752
Sequence   FASTA
 
Membrane scaffold protein 1D1 (deuterated, 75%) (d75-msp1d1)
Mol. type   Protein
Olig. state   Unknown
Mon. MW   24.7 kDa
Sequence   FASTA
 
1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) (d(78,92)-POPC)
Mol. type   Other
Organism   Escherichia coli. (AL95 strain)
Olig. state   Unknown
Mon. MW   0.8 kDa
Sequence   FASTA