The soluble, periplasmic domain of OmpA folds as an independent unit and displays chaperone activity by reducing the self-association propensity of the unfolded OmpA transmembrane β-barrel

Abstract

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 extended conformation, which is in contrast to previous studies that have suggested a collapsed unfolded state.

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

“…Unfolded OMP self-association is another reaction with approximately the same free energy of formation as uOMP/chaperone interactions; for OmpA, this has been estimated to be 29.1 kcal mol 21 measured under conditions similar to those reported for the OMP/chaperone interactions [11,12]. Folding occurs with low efficiency using membranes with partial or full head group properties of E. coli lipids [2,14]. Thus, E. coli lipids are a kinetic barrier to folding and operate as a negative selection against incorporation into bacterial inner membranes.…”

“…Further, it must insert itself into the correct biological membrane, which means that a uOMP nascent chain must traverse through the plasma membrane without folding into it either during or after its bilayer transmittal. And the uOMP must somehow make its way approximately 180 Å across an aqueous periplasmic compartment in which it has marginal-if any-solubility [2]. Only then, when it reaches its outer membrane destination, should the uOMP fold.…”

“…The binding to DegP was slightly slower, on the scale of seconds [13]. Danoff & Fleming [2] showed that uOMPA 171 self-aggregation had an apparent half-life for formation of the order of minutes. Thus, all known interactions in which a uOMP can participate occur Figure 2.…”

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

“…In the case of the OmpA-His3 mutant, a 3xHis-tag was incorporated in an exterior facing transmembrane loop to a truncated form of OmpA which lacked the periplasmic domain which binds to the peptidoglycan layer (Fig. 1A) [22,24,25]. This construct has been utilized by our laboratory previously in combination with a split protein system to direct the lumenal packaging of an active enzyme at either the N-or C-terminus of the recombinant truncated OmpA [26].…”

“…We chose the porin protein OmpA as a scaffold for our construct, as the endogenous protein is highly abundant in bacterial outer membranes, and induction of the mutant OmpA constructs would not prove lethal to the bacterium [20][21][22]. Herein, we demonstrate that both a short 3-histidine (His3) repeat within an exterior protein loop of OmpA and an extended 6-histidine (His6) repeat at the protein terminus can be used for the rapid purification of OMVs.…”