Kinetische Studien über die Faltung und Membraninsertion des Außenmembranproteins A von Escherichia Coli

My work focussed on several significant aspects of the folding mechanism of outer membrane protein A of Escherichia coli, which is composed of a 155 residue periplasmic domain and of a 170 residue transmembrane (TM) domain that forms an 8-stranded TM β-barrel.First, I investigated the folding kinetics of OmpA into model membranes containing the main components of the inner leaflet of the OM: phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The results obtained showed that model membranes mimicking the composition of the outer membrane (containing PE and PG at a molar ratio of 80 and 20) resulted in very low OmpA folding efficiency possibly due to the strong surface-dehydration caused by intermolecular hydrogen-bonds between the ammonium- and the phosphate- parts of PE headgroups. In contrast model membranes where PE was excluded or partially replaced with PC (which forms bilayers and contains a charged trimethyl ammonium group which cannot participate in hydrogen bonding) resulted in high folding yields. When PG was included in moderate amounts (20-30%) into PC/PG membranes, the folding kinetics of OmpA were stimulated significantly because the repulsion between negatively charged PG molecules and the increased hydration shell of this headgroup leads to more water in the headgroup region of negatively charged PG compared to PC.My next two projects sought to explore possible chaperone assisted folding pathways of OmpA that may exist in bacteria. In bacteria, outer membrane proteins like OmpA are synthesized in the cytosol. They are then translocated across the cytoplasmic (inner) membrane into the periplasm in unfolded form. Periplasmic chaperones like SurA or Skp bind to the polypeptide chains after they emerge from the translocon.I demonstrated that OmpA binds ~ 3 molecules of Skp and forms a soluble complex in which OmpA is kept largely unfolded. This complex then binds a small number of n = 2-7 LPS per OmpA in solution to form a folding and insertion component form of OmpA that is bound to Skp and LPS. In this second complex, OmpA develops no or only very small amounts of secondary structure. When this insertion competent form of OmpA was reacted with preformed phospholipid bilayers, OmpA rapidly inserted and folded to its native state. The results indicated that OmpA that is bound to Skp and LPS does not fold in absence of lipid bilayers, but folds into lipid bilayers with accelerated folding kinetics. In contrast, the kinetics of OmpA folding into lipid bilayers from a state bound to Skp in absence of LPS or from a denatured state in 8 M urea were both slower.The SurA assisted folding pathway of OmpA is markedly different in comparison with the folding pathway described above. According to my results, LPS is not required and the presence of SurA prevents the interaction of OmpA with LPS. The chaperone effect of SurA was manifested in experiments where OmpA was incubated in aqueous buffer either in absence or in presence of SurA. In absence of SurA, preincubation in aqueous solution led to aggregated forms of OmpA that did not fold upon subsequent addition of lipid bilayers. In presence of SurA, the formation such aggregated forms was suppressed. SurA therefore appeared to prevent aggregation of OmpA, which still folded into lipid bilayers in high yields. In contrast to experiments with Skp and LPS, for which folding was stimulated compared to simple refolding experiments of urea-denatured OmpA into lipid bilayers, the effect of SurA on unfolded OmpA was small.The last chapter of my thesis presents relevant new data concerning the mechanism of formation and membrane insertion of transmembrane β-barrel domain of OmpA. The goal of my study was to investigate the formation of the transmembrane β-barrel domain of OmpA (residues 1 to 171) on the level of individual β-strands and to relate their association to the insertion of OmpA into the lipid membrane.A new method was developed to monitor the association of individual β-strands in pairs during the formation of the β-barrel domain. I used a series of single Trp, single Cys mutants of OmpA. The Trp and Cys residues of each mutant were located on neighbouring β-strands. The Cysteine-residue was labelled with a nitroxyl spin-label that functions as a short-range fluorescence quencher. Association of the neighboring β-strands during OmpA folding triggers the quenching of Trp fluorescence signal by the spin-labelled Cys residue.The results of the last chapter of the present thesis led to the following conclusions: (i) the assembly of individual strands in pairs during the OmpA barrel formation is a correlated, highly concerted process (and not a sequential one), (ii) the association of individual β-strands in pairs and the sealing of the 8 stranded β-barrel between strands 1 and 8 take place simultaneously (iii) the mechanism of formation and insertion of OmpA β-barrel domain into the lipid bilayers is concerted