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The noncovalent association of transmembrane ␣-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 ToxRmediated 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.The environment presented by the lipid bilayer imposes substantial constraints on the structures of the transmembrane segments of integral membrane proteins, providing a thermodynamic rationale for the formation of stable transmembrane ␣-helices. The establishment of tertiary and quaternary structure then comprises interactions between preformed helical transmembrane domains (tms) (1). However, the study of helix-helix association in the folding of integral membrane proteins is technically difficult because of the necessity for solubilizing membranes or detergent micelles. Here, we present a method to investigate transmembrane helix association in a biological membrane.Although the environment's influence on secondary structure formation is well conceptualized, less is known about the forces that stabilize interactions between transbilayer ␣-helices. Transmembrane helix interactions are governed by the formation of helix-helix contacts and by interactions between the protein and its lipid environment. These environmental influences on folding are poorly understood because of the inability of most experimental systems to directly report helixhelix interactions in their native environment, a natural membrane. Typically, folding studies of membrane proteins have used detergents to provide a convenient membrane-like environment, although the extent to which observations made in detergent micelles accurately reflect helix-he...

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures. The artificial proteins show thermodynamic stabilities similar to natural WW domains, and structure determination of one artificial protein shows excellent agreement with the WW fold at atomic resolution. The relative simplicity of the information used for creating sequences suggests a marked reduction to the potential complexity of the protein-folding problem.