The influence of amino substitutions within the mature part of an Escherichia coli outer membrane protein (OmpA) on assembly of the polypeptide into its membrane.
The membrane part of the 325-residue outer membrane protein OmpA of Escherichia coli encompasses residues 1-177. This part is thought to cross the membrane eight times in antiparallel beta-strands, forming four loops of an amphipathic beta-barrel. With the aim of gaining some insight into the mechanism of sorting, i.e. the way the protein recognizes and assembles into its membrane, a set of point mutants in the ompA gene has been generated. Selection for toxicity of ompA expression following mutagenesis with sodium bisulfite yielded genes with multiple base pair substitutions, the majority of which resulted in amino acid substitutions in the membrane moiety of the protein. None of the altered proteins was blocked in membrane incorporation. A proline residue exists at or near each of the presumed turns at the inner side of the outer membrane. Using oligonucleotide-directed mutagenesis, each of them was replaced by a leucine residue which is thought to be a turn blocking residue. None of these proteins had lost the ability to be incorporated into the membrane. Apparently, leucine residues are tolerated at turns in this protein. To interfere with the formation of antiparallel beta-strands, four double mutants were prepared: ompA-ON3 (Ala11----Pro, Leu13----Pro), -ON4 (Ala11----Asp, Leu13----Pro), -ON5 (Gly160----Val, Leu162----Arg), and -ON6 (Leu164----Pro, Val166----Asp). The former three proteins and even quadruple mutants consisting of a combination of ompA-ON2 or -ON4 with -ON5 were not defective in membrane assembly. In contrast, the OmpA-ON6 protein was translocated across the plasma membrane but could not be incorporated into the outer membrane. It is concluded that at least one rather small area of the polypeptide is of crucial importance for the assembly of OmpA into the outer membrane.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D004926:Escherichia coli / Q000032:analysis*; D006358:Hot Temperature; D008566:Membranes / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000032:analysis*; D010449:Peptide Mapping; D011487:Protein Conformation
D000596:Amino Acids; D009943:Organophosphorus Compounds; C008705:octamethyl pyrophosphoramide