Incorporation of a Rigid TOAC Spin-Label as a Non-Native Amino Acid into a Full-Length Protein by In Vitro Translation using Amber Codon Suppression

Abstract

The development of EPR site-directed spin-labeling (SDSL) techniques has provided a powerful avenue for the study of membrane protein structure and dynamics. One of the advantages of EPR is the ability to obtain distance and dynamic information about particular protein regions. The accuracy of this information is highly dependent on the spin-label that is used. Labels with less inherent mobility are better reporters of protein backbone dynamics and give more sharply defined distance parameters. TOAC is a commonly used rigid spin-label that can be integrated into the protein backbone as an amino acid analog. However, its current use is limited to the study of small peptides that can be only generated synthetically (solid-phase peptide synthesis). A new biochemical approach for incorporation of non-natural amino acids during translation using a synthetically loaded amber suppressor tRNA has successfully generated TOAC-labeled protein but not in sufficient yields for EPR studies. Here, we describe the use of an amber codon suppressor tRNA and a continuous exchange cell-free (CECF) expression system for in vitro translation of a small thylakoid membrane protein, Tha4, containing site-specific integration of TOAC. Further, we demonstrate successful incorporation of the TOAC-containing Tha4 into liposomes by supplying POPC vesicles during the translation reaction. By combining new techniques in suppressor tRNA and continuous exchange cell-free (CECF) translation, we have developed a method for translation of full length proteins with site-directed insertion of non-natural amino acid spin labels in yields applicable to CW-EPR experiments.