Abstract
Here is a novel procedure for making a membrane protein sample, Lipoprotein Signal Peptidase (LspA) for structural studies by solid state NMR. LspA is expressed in E.Coli, purified and refolded with detergent on a Ni-NTA affinitive column and uniformly aligned in lipid bilayer on glass slides. Final concentrations of reconstituted LspA of up to 35mg/mL have been achieved. Reconstitution of LspA in detergent micelles was monitored by CD and solution NMR HSQC. The aligned LspA in lipid bilayer was monitored by solid state NMR to indicate the degree of alignment. Based on these results, ssNMR spectrum will be obtained to show resonance patterns known as PISA wheel for the transmembrane domains of LspA.
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