Abstract
A membrane-spanning heptad repeat motif mediates interaction between transmembrane segments. This motif was randomized with three different sets of mostly hydrophobic residues in the context of POSSYCCAT, a modified ToxR transcription activator system. The resulting combinatorial libraries were subjected to different levels of selective pressure to obtain groups of transmembrane segments that are distinguished by their ability to self-interact in bacterial membranes. Upon relating self-interaction to amino acid composition, the following conclusions were made. First, randomization with only Leu, Ile, Val, Met, and Phe resulted in unexpected robust self-interaction with little sequence specificity. Second, with more complex amino acid mixtures that represent natural transmembrane segments more closely, self-interaction critically depended on amino acid composition of the interface. Whereas the contents of Ile and Leu residues increased with the ability to self-interact, the contents of Pro and Arg residues decreased. Third, heptad repeat motifs composed of Leu, Ile, Val, Met, and Phe were approximately 40-fold over-represented in transmembrane segments of single-span membrane proteins as compared with motifs composed of the more complex amino acid mixtures. This suggests that heptad motifs composed of the smaller subset of amino acids were enriched in the course of natural single-span membrane protein evolution.
Highlights
(a..de.g) pattern of amino acids that is characteristic of soluble leucine zipper interaction domains [8]
The predicted transmembrane segments of single-span or multispan membrane proteins contained in Swiss-Prot version 39ϩ were searched with the XX..XX.XX..XX.XX heptad repeat motif where X corresponds to any amino acid used for transmembrane segment randomization of libraries III, IV, or V; dots correspond to any amino acid except the charged residues lysine, arginine, glutamate, and aspartate
A Regulatable Expression System for ToxR Chimeric Proteins—Chimeric ToxR proteins consist of the cytoplasmic ToxR domain, a transmembrane segment of choice, and a periplasmic maltose binding protein (MalE) domain [20]
Summary
Plasmid Constructs pToxRIV—The ToxR/MalE coding region from vector pToxR⌬TM [13] was amplified using a sense primer with an XbaI site, the ribosome binding site from pASK111 [18], and an antisense primer, including a sequence encoding the myc antibody tag and an XhoI site. The resulting PCR fragment was inserted into pASK111 cut with XbaI/XhoI Upon digesting this vector with XbaI/AseI, the fragment harboring the ToxR/ MalE coding region, the F1 origin, and the chloramphenicol resistance gene was isolated. In the resulting vector the BamHI site of the araBAD promoter was eliminated by point mutagenesis Upon digesting this vector with PstI/ AseI, the fragment harboring the araBAD promoter, the ToxR/MalE coding region, and the araC gene was isolated. This fragment was ligated to a PCR fragment harboring the kanamycin resistance gene and the F1 origin, which was amplified from vector pET24d (Novagen) using a sense primer with the T2 transcription terminator from the cat gene from Staphylococcus aureus plus an NdeI site and an antisense primer with a PstI site.
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