Dataset Information

Differential bacterial surface display of peptides by the transmembrane domain of OmpA.

ABSTRACT: Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor beta-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from 'aberrant' to normal, and predict that some beta-barrels will not display any significant heat-modifiability at all.

SUBMITTER: Verhoeven GS

PROVIDER: S-EPMC2726941 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Differential bacterial surface display of peptides by the transmembrane domain of OmpA.

Verhoeven Gertjan S GS Alexeeva Svetlana S Dogterom Marileen M den Blaauwen Tanneke T

PloS one 20090825 8

Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-termi ...[more]

PMID: 19707582

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Project description:OmpA is one of only a few transmembrane proteins whose folding and stability have been investigated in detail. However, only half of the OmpA mass encodes its transmembrane β-barrel; the remaining sequence is a soluble domain that is localized to the periplasmic side of the outer membrane. To understand how the OmpA periplasmic domain contributes to the stability and folding of the full-length OmpA protein, we cloned, expressed, purified and studied the OmpA periplasmic domain independently of the OmpA transmembrane β-barrel region. Our experiments showed that the OmpA periplasmic domain exists as an independent folding unit with a free energy of folding equal to -6.2 (±0.1) kcal mol(-1) at 25°C. Using circular dichroism, we determined that the OmpA periplasmic domain adopts a mixed alpha/beta secondary structure, a conformation that has previously been used to describe the partially folded non-native state of the full-length OmpA. We further discovered that the OmpA periplasmic domain reduces the self-association propensity of the unfolded barrel domain, but only when covalently attached (in cis). In vitro folding experiments showed that self-association competes with β-barrel folding when allowed to occur before the addition of membranes, and the periplasmic domain enhances the folding efficiency of the full-length protein by reducing its self-association. These results identify a novel chaperone function for the periplasmic domain of OmpA that may be relevant for folding in vivo. We have also extensively investigated the properties of the self-association reaction of unfolded OmpA and found that the transmembrane region must form a critical nucleus comprised of three molecules before undergoing further oligomerization to form large molecular weight species. Finally, we studied the conformation of the unfolded OmpA monomer and found that the folding-competent form of the transmembrane region adopts an expanded conformation, which is in contrast to previous studies that have suggested a collapsed unfolded state.

Dataset Information

Differential bacterial surface display of peptides by the transmembrane domain of OmpA.

Publications

Differential bacterial surface display of peptides by the transmembrane domain of OmpA.

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