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Proc. Natl. Acad. Sci. USAVol. 96, pp. 863–868, February 1999BiochemistryTOXCAT: A measure of transmembrane helix association in abiological membraneWILLIAM P. RUSS AND DONALD M. ENGELMAN*Department of Molecular Biophysics and Biochemistry, Yale University, 420 Bass, 266 Whitney Avenue, P.O. Box 208114, New Haven, CT 06520-8114Contributed by Donald M. Engelman, December 8, 1998ABSTRACT The noncovalent association of transmem-branea-helices is a fundamental event in the folding of helicalmembrane proteins. In this work, a system (TOXCAT) isdeveloped for the study of transmembrane helix– helix oli-gomerization in a natural membrane environment. This assayuses a chimeric construct composed of the N-terminal DNAbinding domain of ToxR (a dimerization-dependent transcrip-tional activator) fused to a transmembrane domain (tm) ofinterest and a monomeric periplasmic anchor (the maltosebinding protein). Association of the tms results in the ToxR-mediated activation of a reporter gene encoding chloramphen-icol acetyltransferase (CAT). The level of CAT expressionindicates the strength of tm association. The assay distin-guishes between a known dimerizing tm and a mutant in whichdimerization is disrupted. In addition, modulation of thechimera concentration shows that the dimerization exhibitsconcentration dependence in membranes. TOXCAT also isused to select oligomeric tms from a library of randomizedsequences, demonstrating the potential of this system to revealnovel oligomerization motifs. The TOXCAT system has beenused to investigate glycophorin A tm-mediated dimerization.Although the overall sensitivity of glycophorin A tm dimer-ization to mutagenesis is found to be similar in membranesand in detergent micelles, several significant differences exist.Mutations to polar residues, which are generally disruptive inSDS, exhibit sequence specificity in membranes, demonstrat-ing both the limitations of detergent micelles and the widerrange of application of the TOXCAT system.The environment presented by the lipid bilayer imposes sub-stantial constraints on the structures of the transmembranesegments of integral membrane proteins, providing a thermo-dynamic rationale for the formation of stable transmembranea-helices. The establishment of tertiary and quaternary struc-ture then comprises interactions between preformed helicaltransmembrane domains (tms) (1). However, the study ofhelix–helix association in the folding of integral membraneproteins is technically difficult because of the necessity forsolubilizing membranes or detergent micelles. Here, wepresent a method to investigate transmembrane helix associ-ation in a biological membrane.Although the environment’s influence on secondary struc-ture formation is well conceptualized, less is known about theforces that stabilize interactions between transbilayera-heli-ces. Transmembrane helix interactions are governed by theformation of helix–helix contacts and by interactions betweenthe protein and its lipid environment. These environmentalinfluences on folding are poorly understood because of theinability of most experimental systems to directly report helix–helix interactions in their native environment, a natural mem-brane. Typically, folding studies of membrane proteins haveused detergents to provide a convenient membrane-like en-vironment, although the extent to which observations made indetergent micelles accurately reflect helix–helix interactions inlipid membranes is not known.The dimerization of the glycophorin A (GpA) tm in deter-gent micelles (2) provides a convenient example of membraneprotein folding. Site-directed mutagenesis (3), computationalmodeling (4), and solution NMR (5) have demonstrated thatthe association between GpAtm monomers is mediated byhelix–helix contacts involving a seven-residue motif, presentedon one face of each transmembranea-helix. The dimerinterface is characterized by tightly packed surfaces formed bycomplementary ridges and grooves that allow close approachof the helices at a right-handed crossing angle (5). Thespecificity of the interaction is such that seemingly conserva-tive mutations of the side chains contributing to the interfacecan disrupt the dimer, whereas hydrophobic mutations atnoninterfacial positions generally have no effect (3, 6). TheGpAtm dimerization motif is sufficient to drive the associationof helices in a detergent environment, even when all nonin-terfacial residues are mutated to leucine (7). In addition, apeptide corresponding to the GpAtm dimerizes in syntheticlipid bilayers (8). Although the energy terms contributed bythe GpAtm helix dimer contacts have been studied in deter-gent micelles (3, 5, 6, 9), little is known about the influence ofenvironment on transmembrane helix–helix association.To study tm association in a natural membrane environ-ment, we have developed the TOXCAT assay system, which isbased on the dimerization-dependent ToxR transcriptionalactivation domain (10, 11). TOXCAT provides substantialadvantages over previous implementations of ToxR (11, 12),exhibiting heightened sensitivity to changes in dimerizationaffinity, tunable expression level, and the ability to applyselective pressure to isolate strongly oligomerizing tms. Ap-plication of this system to investigate the effects of mutagenesisof the GpAtm dimerization domain in a natural membrane hasdemonstrated significant environmental influences on theassociation of transmembranea-helices.MATERIALS AND METHODSGeneral. Media were prepared as described in Sambrook etal. (13). All genes cloned using PCR were confirmed bysequence analysis. Ampicillin (Amp) was used at 200mgyml.Vectors and Constructs. pMAL-c2 and –p2 were obtainedfrom New England Biolabs. The ctx::chloramphenicol acetyl-transferase (CAT) reporter construct was cloned by PCRamplifying the ctx promoter from genomic DNA isolated fromEscherichia coli FHK12 (kindly provided by H. Kolmar,George-August University, Go¨ttingen, Germany; ref. 11),using primers that add HindIII sites at both termini. Theresulting product was ligated into the HindIII site of pkk232–8The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked ‘‘advertisement’’ inaccordance with 18 U.S.C. §1734 solely to indicate this fact.PNAS is available online at www.pnas.org.Abbreviations: CAT, chloramphenicol acetyltransferase; MBP, mal-tose binding protein; GpA, glycophorin A; tm, transmembrane


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