The eight-stranded antiparallel beta-barrel domain of the OmpA protein from Escherichia coli serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. Previous studies have shown that neither the periplasmic turns nor the surface-exposed loops contain topogenic information. Consequently, the question of whether any structural constraint is imposed onto individual transmembrane beta-strands is now addressed. To this end, amino acid sequences of beta-strands 4, 6, and 8 were randomized. In vivo membrane assembly of mutant proteins was assayed and 288 variants were sequenced. Three parameters were found to be important for efficient membrane assembly: (i) At least four of five randomized residues with side chains pointing towards the lipid bilayer must be hydrophobic and none of the three central residues must be charged. (ii) Side chains pointing into the beta-barrel interior must not be enlarged to much, possibly because of packing constraints. (iii) Proline residues are, in general, hardly tolerated in the transmembrane beta-strands.
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