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.
Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature
From The DNP-NMR Blog:
Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature
Solid-state DNP-NMR are typically performed at cryogenic temperatures and samples, especially bio-macromolecules often require cryo-protection. This is a recent review about sample preparation and cryo-protecting samples to preserve the spectral resolution.
Lee, M. and M. Hong, Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature. J Biomol NMR, 2014....
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08-27-2014 02:29 PM
[NMR paper] Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature.
Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature.
Related Articles Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature.
J Biomol NMR. 2014 Jul 12;
Authors: Lee M, Hong M
Abstract
Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the...
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07-13-2014 06:48 PM
Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature
Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature
Abstract
Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200Â*K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are...
[NMR paper] Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR.
Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR.
Related Articles Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR.
Biochemistry. 2014 Mar 25;
Authors: Murray DT, Griffin JM, Cross TA
Abstract
For small helical membrane proteins their structure is highly sensitive to their environment and solid state NMR is a structural technique that can characterize these membrane proteins in native like lipid bilayers and proteoliposomes. To date, a systematic method by which to...
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03-29-2014 01:00 PM
[NMR paper] Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals to
Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with amino acid residues.
Related Articles Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with amino acid residues.
Magn Reson Chem. 2004 Feb;42(2):218-30
Authors: Saitô H, Mikami J, Yamaguchi S, Tanio M, Kira A, Arakawa T, Yamamoto K, Tuzi S
We have so...
Gradient reconstitution of membrane proteins for solid-state NMR studies
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