Gradient reconstitution of membrane proteins for solid-state NMR studies.
J Biomol NMR
; 69(2): 81-91, 2017 Oct.
Article
en En
| MEDLINE
| ID: mdl-28900789
ABSTRACT
We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution, and shall represent a valuable alternative in solid-state NMR membrane protein sample preparation.
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Banco de datos:
MEDLINE
Asunto principal:
Resonancia Magnética Nuclear Biomolecular
/
Proteínas de la Membrana
Idioma:
En
Año:
2017
Tipo del documento:
Article