One β Hairpin Follows the Other: Exploring Refolding Pathways and Kinetics of the Transmembrane β‐Barrel Protein OmpG

Damaghi, Mehdi; Köster, Stefan; Bippes, Christian A.; Yıldız, Özkan; Müller, Daniel J.

doi:10.1002/ange.201101450

Cited by 12 publications

(5 citation statements)

References 20 publications

(24 reference statements)

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“…As expected from previous studies, [7a, 8b, 12] loop 6 performs the major gating function. When it was pinned to the membrane, a constitutively open pore was found with properties quite similar to those of quietOmpG (Figure S3).…”

supporting

confidence: 85%

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

Zhuang¹,

Tamm²

2014

Angew Chem Int Ed

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Nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms nanopores from single polypeptide chains. This property affords one to separately engineer each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized previously as the main gating loop that closes the pore at low pH and opens it at high pH. In the present work, we devised a method to pin each of these loops to the embedding membrane and measure the single pore conductances of these constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighbouring and distant loops in a correlated fashion. Pinning of loop 6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore.

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confidence: 85%

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

Zhuang¹,

Tamm²

2014

Angew Chem Int Ed

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“…In the case of transmembrane a-helical proteins, it was shown that the mechanically stressed polypeptide not only induces the stepwise unfolding of the membrane protein but also folds back stepwise into the membrane bilayer (Kedrov et al, 2006;Kedrov et al, 2004;Kessler et al, 2006). Similarly, mechanically loaded OmpG unfolds stepwise and refolds stepwise into the membrane (Damaghi et al, 2011). Here we show that when mechanically stressing the PGBD of KpOmpA, transmembrane b-strands form stable unfolding intermediates and, as soon as the tensile load disappears, fold back into the lipid membrane.…”

Section: Possible Extension Of the Two-state Folding And Insertion Momentioning

confidence: 99%

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

Bosshart¹,

Iordanov²,

Garzón-Coral³

et al. 2012

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In Klebsiella pneumoniae the transmembrane β-barrel forming outer membrane protein KpOmpA mediates adhesion to a wide range of immune effector cells, thereby promoting respiratory tract and urinary infections. As major transmembrane protein OmpA stabilizes Gram-negative bacteria by anchoring their outer membrane to the peptidoglycan layer. Adhesion, osmotic pressure, hydrodynamic flow, and structural deformation apply mechanical stress to the bacterium. This stress can generate tensile load to the peptidoglycan-binding domain (PGBD) of KpOmpA. To investigate how KpOmpA reacts to mechanical stress, we applied a tensile load to the PGBD and observed a detailed unfolding pathway of the transmembrane β-barrel. Each step of the unfolding pathway extended the polypeptide connecting the bacterial outer membrane to the peptidoglycan layer and absorbed mechanical energy. After relieving the tensile load, KpOmpA reversibly refolded back into the membrane. These results suggest that bacteria may reversibly unfold transmembrane proteins in response to mechanical stress.

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“…Such mechanical stress may be expected when bacteria adhering to surfaces are exposed to hydrodynamic flow or undergo severe mechanical deformations (Otto et al, 2001). It has also been shown that b barrel proteins once unfolded by mechanical force can insert and fold back into the lipid membrane (Bosshart et al, 2012a;Damaghi et al, 2011). Thereby the unfolded polypeptide can insert one b hairpin after the other until folding of the b barrel has been completed.…”

Section: Introductionmentioning

confidence: 99%

“…In the latter case, periplasmic chaperones can prevent misfolding and support the stepwise insertion and folding of b hairpins toward the native membrane protein structure (Thoma et al, 2015). Thus, studying the mechanical unfolding of b barrel proteins can provide complementary mechanistic insight into their folding behavior (Bosshart et al, 2012a;Damaghi et al, 2011;Horne and Radford, 2016;Thoma et al, 2015). In previous studies we used AFM-based SMFS to investigate the mechanical unfolding of bacterial outer membrane proteins having different sizes: (1) the small outer membrane protein OmpA from Klebsiella pneumoniae comprising eight b strands (Bosshart et al, 2012a), (2) the intermediate-sized outer membrane protein OmpG from Escherichia coli comprising 14 b strands (Sapra et al, 2009), and (3) the large outer membrane protein FhuA from E. coli comprising 22 b strands (Thoma et al, 2012) Here we attempt to gain a deeper understanding of the mechanical unfolding pathways of outer membrane proteins observed so far and apply SMFS to unfold another b barrel membrane protein maltoporin (LamB) from E. coli.…”

Section: Introductionmentioning

confidence: 99%

Maltoporin LamB Unfolds β Hairpins along Mechanical Stress-Dependent Unfolding Pathways

et al. 2017

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Upon mechanical pulling at either terminal end, β barrel outer membrane proteins stepwise unfold β strands or β hairpins until entirely extracted from the membrane. This unique unfolding pathway has been described for β barrels comprising 8, 14, or 22 β strands. Here we mechanically unfold the 18-stranded β barrel outer membrane protein LamB from Escherichia coli. We find that its mechanical unfolding pathway is shaped by the stepwise unfolding of β hairpins. However, we also observe that β hairpins can unfold groupwise. Thereby, β hairpins unfolding at higher pulling forces show a higher probability to unfold collectively, whereas β hairpins unfolding at lower forces tend to unfold individually. This result suggests that the collective unfolding of β hairpins resembles a far-from-equilibrium process, whereas the unfolding of individual β hairpins describes a closer-to-equilibrium process. Our findings support a direct link between outer membrane protein structure and the unfolding pathway and contribute to a better understanding of their unfolding in response to mechanical stress.

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One β Hairpin Follows the Other: Exploring Refolding Pathways and Kinetics of the Transmembrane β‐Barrel Protein OmpG

Cited by 12 publications

References 20 publications

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

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

Maltoporin LamB Unfolds β Hairpins along Mechanical Stress-Dependent Unfolding Pathways

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