Whole-cell circular dichroism difference spectroscopy reveals an in vivo-specific deca-heme conformation in bacterial surface cytochromes

We established whole-cell circular dichroism difference spectroscopy to identify the inter-heme interaction in deca-heme cytochrome protein MtrC in whole cell. Our data showed that the heme alignment of reduced MtrC in whole cell is distinct from that in purified one, suggesting the in vivo specific electron transport kinetics

Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain

Skp is a multivalent chaperone of outer membrane proteins

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry–mass spectrometry (IMS–MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation

Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date?

Research into the mechanisms by which proteins fold into their native structures has been on-going since the work of Anfinsen in the 1960s. Since that time, the folding mechanisms of small, water-soluble proteins have been well characterised. By contrast, progress in understanding the biogenesis and folding mechanisms of integral membrane proteins has lagged significantly because of the need to create a membrane mimetic environment for folding studies in vitro and the difficulties in finding suitable conditions in which reversible folding can be achieved. Improved knowledge of the factors that promote membrane protein folding and disfavour aggregation now allows studies of folding into lipid bilayers in vitro to be performed. Consequently, mechanistic details and structural information about membrane protein folding are now emerging at an ever increasing pace. Using the panoply of methods developed for studies of the folding of water-soluble proteins. This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information. The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins

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

Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide

We have studied the folding pathway of a β-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA

Association of neighboring β-strands of outer membrane protein A in lipid bilayers revealed by site-directed fluorescence quenching

We present a detailed study on the formation of neighboring β-strands during the folding of a monomeric integral membrane protein of the β-barrel type. β-Strand and β-barrel formations were investigated for the eight-stranded transmembrane domain of outer membrane protein A (OmpA) with single-tryptophan (W), single-cysteine (C) OmpA mutants. Based on the OmpA structure, W and C were introduced in two neighboring β-strands oriented toward the hydrocarbon core of the membrane. Replaced residue pairs were closer to either the periplasmic turns (named cis-side) or the outer loops (named trans-side) of the strand. WnCm OmpA mutants containing W at position n and C at position m along the polypeptide chain were labeled at the C by a nitroxyl spin label, which is a short-range fluorescence quencher. To monitor the association of neighboring β-strands, we determined the proximity between fluorescent W and labeled C in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native β-strand contacts in folding experiments required the lipid membrane. Residues in the trans-side of strands β1, β2, and β3, represented by mutants W15C35 (β1β2, trans) and W57C35 (β3β2, trans), reached close proximity prior to residues in the N(β1)- and C(β8)-terminal strands as examined for mutants W15C162 (β1β8, trans) and W7C170 (β1β8, cis). Tryptophan and cysteine converged slightly faster in W15C162 (β1β8, trans) than in W7C170 (β1β8, cis). The last folding step was observed for residues at the cis-ends of strands β1 and β2 for the mutant W7C43 (β1β2, cis). The data also demonstrate that the neighboring β-strands associate upon insertion into the hydrophobic core of the lipid bilayer

Data from: Structure of FlgK reveals the divergence of the bacterial hook-filament junction of Campylobacter

Evolution of a nano-machine consisting of multiple parts, each with a specific function, is a complex process. A change in one part should eventually result in changes in other parts, if the overall function is to be conserved. In bacterial flagella, the filament and the hook have distinct functions and their respective proteins, FliC and FlgE, have different three-dimensional structures. The filament functions as a helical propeller and the hook as a flexible universal joint. Two proteins, FlgK and FlgL, assure a smooth connectivity between the hook and the filament. Here we show that, in Campylobacter, the 3D structure of FlgK differs from that of its orthologs in Salmonella and Burkholderia, whose structures have previously been solved. Docking the model of the FlgK junction onto the structure of the Campylobacter hook provides some clues about its divergence. These data suggest how evolutionary pressure to adapt to structural constraints, due to the structure of Campylobacter hook, causes divergence of one element of a supra-molecular complex in order to maintain the function of the entire flagellar assembly