In vitro selection of membrane-spanning leucine zipper protein-protein interaction motifs using POSSYCCAT

A membrane-spanning heptad repeat motif mediates interaction between transm embrane segments. This motif was randomized with three different sets of mo stly hydrophobic residues in the context of POSSYCCAT, a modified ToxR tran scription activator system. The resulting combinatorial libraries were subj ected to different levels of selective pressure to obtain groups of transme mbrane segments that are distinguished by their ability to self-interact in bacterial membranes. Upon relating self-interaction to amino acid composit ion, the following conclusions were made. First, randomization with only Le u, Ile, Val, Met, and Phe resulted in unexpected robust self-interaction wi th little sequence specificity. Second, with more complex amino acid mixtur es that represent natural transmembrane segments more closely, self-interac tion critically depended on amino acid composition of the interface. Wherea s the contents of Ile and Leu residues increased with the ability to self-i nteract, the contents of Pro and Arg residues decreased. Third, heptad repe at motifs composed of Leu, Ile, Val, Met, and Phe were similar to 40-fold o ver-represented in transmembrane segments of single-span membrane proteins as compared with motifs composed of the more complex amino acid mixtures. T his suggests that heptad motifs composed of the smaller subset of amino aci ds were enriched in the course of natural single-span membrane protein evol ution.