MEMBRANE TOPOLOGY AND ASSEMBLY OF THE OUTER-MEMBRANE PROTEIN OMPA OF ESCHERICHIA-COLI K12

The 325-residue outer membrane protein OmpA of Escherichia coli has be en proposed to consist of a membrane-embedded moiety (residues 1 to ab out 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel bet a-strands, forming an amphiphilic beta-barrel, connected by exposed tu rns. Several questions concerning this model were addressed. Thus no e xperimental evidence had been presented for the turns at the inner lea flet of the membrane and it was not known whether or not the periplasm ic part of the polypeptide plays a role in the process of membrane inc orporation. Oligonucleotides encoding trypsin cleavage sites were inse rted at the predicted turn sites of the ompA gene and it was shown tha t the encoded proteins indeed become accessible to trypsin at the modi fied sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically to pogenic information. In two cases one of the two expected tryptic frag ments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes ca n cause loss of beta-strands and does not necessarily produce a reliab le picture of protein topology. When ompA genes were constructed codin g for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of t he proteins not being assembled. No such defect was observed when, fol lowing the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the pr oposed beta-barrel structure is essentially correct and (2) the peripl asmic part of OmpA does not play an active role in, but can, when pres ent in mutant form, interfere with membrane assembly.