The Transmembrane Protein KpOmpA Anchoring the Outer Membrane of Klebsiella pneumoniae Unfolds and Refolds in Response to Tensile Load

Bosshart, Patrick D.; Iordanov, I; Garzón-Coral, Carlos; Demange, Pascal; Engel, Andréas; Milon, Alain; Müller, Daniel J.

doi:10.1016/j.str.2011.11.002

Cited by 36 publications

(50 citation statements)

References 50 publications

(88 reference statements)

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“…It was previously shown, that each force peak of this unfolding pattern describes an unfolding step and intermediate of OmpG 46,48,49 . It was also shown, that this pattern is specific for pulling the N-terminal end of OmpG, which faces the periplasmic space of E. coli 46,48,49 . The OMV membranes thus exposed their periplasmic surface to the buffer solution.…”

Section: Resultsmentioning

confidence: 92%

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Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies

et al. 2018

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Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. Whereas solubilized membrane proteins lack the functionally important lipid membrane, reconstitution embeds them into artificial lipid bilayers, which lack characteristic features of cellular membranes including lipid diversity, composition and asymmetry. Here, we utilize outer membrane vesicles (OMVs) released from Escherichia coli to study outer membrane proteins (Omps) in the native membrane environment. Enriched in the native membrane of the OMV we characterize the assembly, folding, and structure of OmpG, FhuA, Tsx, and BamA. Comparing Omps in OMVs to those reconstituted into artificial lipid membranes, we observe different unfolding pathways for some Omps. This observation highlights the importance of the native membrane environment to maintain the native structure and function relationship of Omps. Our fast and easy approach paves the way for functional and structural studies of Omps in the native membrane.

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Section: Resultsmentioning

confidence: 92%

“…For each approach and retraction cycle a force-distance curve was recorded. In about 0.3‰ of attempts ( n = 162,657), the force-distance curves showed the distinct saw-tooth like unfolding pattern of an Omp 46,48,49 . This indicated that the OmpG density in OMVs was sufficient to pick up single OmpG molecules for SMFS.…”

Section: Resultsmentioning

confidence: 99%

Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies

et al. 2018

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“…Furthermore, because these interactions alter the physical properties of LacY (reviewed in ref. 9), the energetic, kinetic, and mechanical properties of LacY that fulfill different functional roles during transport remain to be characterized.Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20)(21)(22)(23)(24)(25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26-35).…”

mentioning

confidence: 99%

“…Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20)(21)(22)(23)(24)(25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”

mentioning

confidence: 99%

Substrate-induced changes in the structural properties of LacY

Serdiuk

Madej²,

Sugihara³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugarand H + -binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H + -and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H + symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.atomic force microscopy | membrane | transport protein | membrane protein structure | membrane protein folding | membrane transport T he lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + (sugar/H + symport) (3-6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H + with the electrochemical H + gradient (Δμ H + ; interior negative and/or alkaline). Conversely, because coupling between sugar and H + is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H + transport with the generation of Δμ H +, the polarity of which depends upon the direction of the sugar concentration gradient (7-10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13-16). At the apex of the h...

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“…(19). While the refolding pathway of chemically denatured OmpA protein appears to be best described by the concerted process, it is interesting to note that mechanical unfolding of the related protein KpOmpA from Klebsiella pneumoniae results in a stepwise unfolding process where individual β hairpins unfold successively [64]. Similar behavior has also been observed in β barrels OmpG and FhuA [65,66].…”

Section: Folding Pathways For β Barrelsmentioning

confidence: 70%

Model for coupled insertion and folding of membrane-spanning proteins

Hausrath

2014

Phys. Rev. E

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Current understanding of the forces directing the folding of integral membrane proteins is very limited compared to the detailed picture available for water-soluble proteins. While mechanistic studies of the folding process in vitro have been conducted for only a small number of membrane proteins, the available evidence indicates that their folding process is thermodynamically driven like that of soluble proteins. In vivo, however, the majority of integral membrane proteins are installed in membranes by dedicated machinery, suggesting that the cellular systems may act to facilitate and regulate the spontaneous physical process of folding. Both the in vitro folding process and the in vivo pathway must navigate an energy landscape dominated by the energetically favorable burial of hydrophobic segments in the membrane interior and the opposition to folding due to the need for passage of polar segments across the membrane. This manuscript describes a simple, exactly solvable model which incorporates these essential features of membrane protein folding. The model is used to compare the folding time under conditions which depict both the in vitro and in vivo pathways. It is proposed that the cellular complexes responsible for insertion of membrane proteins act by lowering the energy barrier for passage of polar regions through the membrane, thereby allowing the chain to more rapidly achieve the folded state.

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The Transmembrane Protein KpOmpA Anchoring the Outer Membrane of Klebsiella pneumoniae Unfolds and Refolds in Response to Tensile Load

Cited by 36 publications

References 50 publications

Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies

Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies

Substrate-induced changes in the structural properties of LacY

Model for coupled insertion and folding of membrane-spanning proteins

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