Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles
Résumé
TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein.
We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, w:w), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased.
Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: Dmax ∼95 Å in DPC alone versus ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (Kd ∼70 μM in DPC alone versus ∼0.91 μM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST).
In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.
Mots clés
dodecyl phosphocholine
MALLS
multi-angle laser light scattering
MST
microscale thermophoresis
mTSPO
mouse TSPO
SANS
smallangle neutron scattering
SAXS
small-angle X-ray scattering
SDS
sodium dodecyl sulfate
SEC
size-exclusion chromatography
SLD
scattering length density
Trp
tryptophan
TSPO
membrane protein
small-angle scattering (SAXS/SANS)
DPC
DMPC
mouse TSPO circular dichroism DDM
dodecyl maltoside DM
decyl maltoside DMPC
1
2 DPC
dodecyl phosphocholine MALLS
multi-angle laser light scattering MST
microscale thermophoresis mTSPO
mouse TSPO SANS
smallangle neutron scattering SAXS
small-angle X-ray scattering SDS
sodium dodecyl sulfate SEC
size-exclusion chromatography SLD
scattering length density Trp
tryptophan TSPO
translocator protein
mouse TSPO circular dichroism
DDM
dodecyl maltoside
DM
decyl maltoside
2
Domaines
Sciences du Vivant [q-bio]Origine | Fichiers produits par l'(les) auteur(s) |
---|