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Nature Protocols 2, 42–49 (1 January 2007) | doi:10.1038/nprot.2007.2

Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes

Christine B Karim , Zhiwen Zhang & David D Thomas

A procedure is described for the synthetic incorporation into membrane proteins of the non-natural amino acid TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid), which is coupled rigidly to the α-carbon, providing direct detection of peptide backbone dynamics by electron paramagnetic resonance (EPR). Also included is a protocol for the functional reconstitution of the spin-labeled protein in lipid vesicles. This protocol can be completed in 17 d.