TOXCAT: A measure of transmembrane helix association in a biological membrane

William P. Russ, Donald M. Engelman

Research output: Contribution to journalArticlepeer-review

309 Scopus citations

Abstract

The noncovalent association of transmembrane α-helices is a fundamental event in the folding of helical membrane proteins. In this work, a system (TOXCAT) is developed for the study of transmembrane helix-helix oligomerization in a natural membrane environment. This assay uses a chimeric construct composed of the N-terminal DNA binding domain of ToxR (a dimerization-dependent transcriptional activator) fused to a transmembrane domain (tm) of interest and a monomeric periplasmic anchor (the maltose binding protein). Association of the tms results in the ToxR-mediated activation of a reporter gene encoding chloramphenicol acetyltransferase (CAT). The level of CAT expression indicates the strength of tm association. The assay distinguishes between a known dimerizing tm and a mutant in which dimerization is disrupted. In addition, modulation of the chimera concentration shows that the dimerization exhibits concentration dependence in membranes. TOXCAT also is used to select oligomeric tms from a library of randomized sequences, demonstrating the potential of this system to reveal novel oligomerization motifs. The TOXCAT system has been used to investigate glycophorin A tm-mediated dimerization. Although the overall sensitivity of glycophorin A tm dimerization to mutagenesis is found to be similar in membranes and in detergent micelles, several significant differences exist. Mutations to polar residues, which are generally disruptive in SDS, exhibit sequence specificity in membranes, demonstrating both the limitations of detergent micelles and the wider range of application of the TOXCAT system.

Original languageEnglish (US)
Pages (from-to)863-868
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number3
DOIs
StatePublished - Feb 2 1999

ASJC Scopus subject areas

  • General

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