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

Nature Protocols volume 2, pages 42–49 (2007)Cite this article

Abstract

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.

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Figure 1: Structural model of AFA-PLB, based on NMR1.
Figure 2: Typical EPR spectrum of resin-bound (Fmoc-PAL-PEG-PS) TOAC spin-labeled protein.
Figure 3: Spectra of sequential synthetic intermediates in lipid bilayers.
Figure 4: At strong acid pH, Tempo (a) disproportionates to oxoammonium cation (b) and TempOH (c).
Figure 5: EPR spectra of a TOAC spin-labeled protein reconstituted in lipid bilayers.
Figure 6: Determination of [Spin]/[PLB].

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Acknowledgements

The authors thank Henriette Remmer at the Department of Biological Chemistry, University of Michigan, for advice about experimental details. This work was supported by a grant from the National Institutes of Health to D.D.T. (GM27906).

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  1. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, 55455, MN, USA

    Christine B Karim, Zhiwen Zhang & David D Thomas

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Correspondence to Christine B Karim.

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Karim, C., Zhang, Z. & Thomas, D. Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes. Nat Protoc 2, 42–49 (2007). https://doi.org/10.1038/nprot.2007.2

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