Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes.
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 alpha-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.
Karim
Christine B
CB
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. cbk@ddt.biochem.umn.edu
Zhang
Zhiwen
Z
Thomas
David D
DD
D002135:Calcium-Binding Proteins / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D013113:Spin Labels / Q000138:chemical synthesis*; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D003497:Cyclic N-Oxides; D008055:Lipids; D013113:Spin Labels; D000099211:Phospholamban; C037109:2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylic acid