Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

Abstract

Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.T he cellular envelope of Gram-negative bacteria is comprised of two membranes separated by an aqueous compartment termed the "periplasm." The outer membrane of the cellular envelope contains integral β-barrel membrane proteins referred to as "outer membrane proteins" (OMPs) (1, 2). The outer membrane and OMPs provide the first barrier between bacteria and the environment and are essential to many important cellular processes including metabolic transport, bacterial virulence, and antibiotic resistance (3-5). Understanding the pathway by which OMPs traverse the periplasm and attain their native functional state is essential to an ability to manipulate this element of the bacterial cell.The OMP biogenesis process is distinct from the folding of cytosolic proteins because it involves a unique collection of obstacles. First, OMPs do not adopt their folded conformations while in an aqueous environment (6). Rather, unfolded OMPs (uOMPs) must be transported across the periplasm to reach their native membrane. Because of their marginal solubility in water, this process must be tightly controlled to avoid aggregation. Second, structures of folded OMPs (fOMPs) show that these proteins contain water-solvated residues in loops on the outer surfaces of bacteria. The desolvation and transport of these polar and ionizable side chains across the outer membrane represent significant kinetic barriers to OMP folding (7). Third, Gram-negative bacteria maintain a high density of fOMP in the expanding oute...

Orientation of the OmpF Porin in Planar Lipid Bilayers

“…The insertion of OmpF into a bilayer with the periplasmic turns first is not observed in the bacterialO M, where assemblyo ft he porin is guided by chaperonesa nd the b-barrel assembly machinery. [36,37] In PLBs, maltoporin (another trimeric E. coli OM protein) hasbeen observed to insert predominantly with the periplasmic side first from diluted octyl-POE at an applied potential of AE (100-200 mV). [28] Our reconstitution protocol similarly uses potentials in the AE (200-300) mV range to insert OmpF from diluted bOG, and it is unclear why we saw both orientationswith equal probability.…”

Novel Kinetic Intermediates Populated along the Folding Pathway of the Transmembrane β-Barrel OmpA

“…While chaperones ensure that nascent OMP chains transit across the aqueous periplasm, insertion into the outer membranes is the final, rate-limiting step in OMP maturation. 58 This reaction is catalyzed by the multi-protein BAM complex in which the central essential subunit, BamA, is itself a transmembrane β-barrel. By its very nature, BamA must have extensive interactions with membrane lipids.…”

High affinity of Skp to OmpC revealed by single-molecule detection

“…The reaction rate of Skp binding OmpC is nearly diffusion limited 30 . The association rate constant could be estimated by…”

“…When OMP polypeptide was synthesized by ribosome in the cytoplasm, the polypeptide would be secreted to periplasm through SecYEG/SecA translocon in unfolded state and safeguarded by chaperones including Skp3 1,26-29 . Many articles showed that OMP×Skp3 has a dissociation constant of nano-molar range19,30,31 . Most their experiments, however, were performed at ensemble level of OMPs,…”