β-Barrel Proteins That Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro

Abstract

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane ␤-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.

Outer Membrane Biogenesis

“…2B) (68). This configuration of the seam was not surprising because it has been known for a long time that the BamA barrel has the lowest thermodynamic stability in comparison to other β-barrels (9). Barrel stability depends in large part on the number of hydrogen bonds formed between β-strands, and this low BamA stability with a melting temperature (Tm) around 37° C is intriguing, because it suggests that the BamA barrel may be highly dynamic at this physiological temperature.…”

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.…”

“…No configuration of natural or mimicked E. coli lipids support fast folding in vitro: neither of the forms of E. coli lipids available for purchase from Avanti support high efficiency folding; nor do the lipids purified from E. coli; nor do bilayers prepared with synthetic lipids mixed at biological mole ratios; and the addition of lipopolysaccharide has no dramatic effect on folding [14,15]. Upon discovery, this was surprising and counterintuitive.…”

Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

“…Reductions in temperature will also further decrease the fluidity of the characteristically rigid OM (15,16), which could exacerbate assembly defects of certain classes of proteins. The kinetics of OMP folding into a membrane is strongly influenced by the physical properties of the membrane, and temperature can strongly affect the biophysical properties of the OM, leading to changes in the rate of OMP folding with temperature (17)(18)(19)(20). Alternatively, or in addition, it seems likely that there is an increased requirement for periplasmic proteolysis in the BamA/BamD down mutant strains.…”