Gradient reconstitution of membrane proteins for solid-state NMR studies.
Related Articles Gradient reconstitution of membrane proteins for solid-state NMR studies.
J Biomol NMR. 2017 Sep 12;:
Authors: Lacabanne D, Lends A, Danis C, Kunert B, Fogeron ML, Jirasko V, Chuilon C, Lecoq L, Orelle C, Chaptal V, Falson P, Jault JM, Meier BH, Böckmann A
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.
PMID: 28900789 [PubMed - as supplied by publisher]
More...