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Gradient reconstitution of membrane proteins for solid-state NMR studies.

Denis LacabanneAlons LendsClément DanisBritta KunertMarie-Laure FogeronVlastimil JiraskoClaire ChuilonLauriane LecoqCédric OrelleVincent ChaptalPierre FalsonJean-Michel JaultBeat H MeierAnja Böckmann
Published in: Journal of biomolecular NMR (2017)
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.
Keyphrases
  • solid state
  • chronic kidney disease
  • electron microscopy
  • molecularly imprinted
  • end stage renal disease
  • fatty acid
  • protein protein
  • case control
  • mass spectrometry
  • peritoneal dialysis
  • capillary electrophoresis