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Proteins, Expression, Isolation and Analysis | … After 1 h of centrifugation at 16,000 × g, mix supernatant with 5 mL of Ni-beads (Genscript) for 1 h at room temperature … 2 Sample preparation and solution NMR spectroscopy of the β-barrel membrane protein BamA … 3.7 Toolbox of MAS-NMR Experiments for Membrane Proteins … | Get A Quote |
NMR spectroscopy is a method of choice to characterize structure, function, and dynamics of integral membrane proteins at atomic resolution. Here, we describe protocols for sample preparation and characterization by NMR spectroscopy of two integral membrane proteins with different architecture, the α-helical membrane protein MsbA and the β-barrel membrane protein BamA. The protocols describe recombinant expression in E. coli, protein refolding, purification, and reconstitution in suitable membrane mimetics, as well as key setup steps for basic NMR experiments. These include experiments on protein samples in the solid state under magic angle spinning (MAS) conditions and experiments on protein samples in aqueo... More