Membrane assembly of the outer membrane protein OmpA of Escherichia coli.

The membrane part (residues 1 to approximately 170) of the 325-residue Escherichia coli outer membrane protein OmpA is thought to exist in the membrane as an 8-stranded beta-barrel, subdividing this part into four segments. The influence of proline residues on membrane assembly of the protein has been studied. These were introduced, using site-directed mutagenesis, into each of seven of the antiparallel beta-strands. One important parameter for allowing or not allowing membrane assembly was the potential H beta (i) which is the potential to form an amphiphilic beta-strand. When H beta (i) remained unaltered, 2 prolines were tolerated. Lowering H beta (i) in most cases caused failure of assembly when 2 such residues were present. An insert of 10 residues, including 3 prolines, did not alter H beta(i) and was tolerated, but caused "looping out" of the strand to the outer face of the membrane; displacement to its inner side would not have allowed for an amphiphilic beta-strand. Thus, a beta-structured protein is as adaptable as it has been shown for an alpha-helix. The wild type segment order 1-2-3-4 has been changed to 1-3-3-4 and 1-4-3-4. Since the proteins were found associated with the outer membrane but could not be incorporated into it, it appears that sorting is less sensitive to alterations than assembly. A regulatory circuit was affected (missense mutants of outer membrane proteins can cause inhibition of synthesis of other such proteins); expression of the two rearranged genes effected a strong inhibition of synthesis of the unrelated porins OmpC and F as well as that of the maltoporin LamB and wild type OmpA. Hence, outer membrane proteins are designed not only for efficient membrane assembly but also for proper regulation of their synthesis.