Expression of correctly folded proteins in Escherichia coli

AbstractCellular proteomes are dynamic and adjusted to permanently changing conditions by ATP-fueled proteolytic machineries. Among the five AAA+proteases inEscherichia coliFtsH is the only essential and membrane-anchored metalloprotease. FtsH is a homohexamer that uses its ATPase domain to unfold and translocate substrates that are subsequently degraded without the need of ATP in the proteolytic chamber of the protease domain. FtsH eliminates misfolded proteins in the context of general quality control and properly folded proteins for regulatory reasons. Recent trapping approaches have revealed a number of novel FtsH substrates. This review summarizes the substrate diversity of FtsH and presents details on the surprisingly diverse recognition principles of three well-characterized substrates: LpxC, the key enzyme of lipopolysaccharide biosynthesis; RpoH, the alternative heat-shock sigma factor and YfgM, a bifunctional membrane protein implicated in periplasmic chaperone functions and cytoplasmic stress adaptation.

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TatA and TatB generate a hydrophobic mismatch that is important for function and assembly of the Tat translocon in Escherichia coli

10.1101/2021.05.26.445790 ◽

2021 ◽

Author(s):

Denise Mehner-Breitfeld ◽

Michael T. Ringel ◽

Daniel Alexander Tichy ◽

Laura J. Endter ◽

Kai Steffen Stroh ◽

...

Keyword(s):

Escherichia Coli ◽

Transmembrane Helix ◽

Length Variation ◽

Hydrophobic Mismatch ◽

Hydrophobic Residues ◽

Transmission Electron ◽

Absolute Requirement ◽

Bacterial Model ◽

Folded Proteins ◽

Dynamics Simulations

The Tat system translocates folded proteins across energy-transducing prokaryotic membranes. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was tolerated. Length-variation in TatB clearly destabilized TatBC-containing complexes, indicating that the 12-residues-length is crucial for Tat component interactions and translocon assembly. Metal tagging transmission electron microscopy revealed the dimensions of TatA assemblies, which prompted molecular dynamics simulations. These showed that interacting TMHs of larger TatA assemblies can thin the membrane together with laterally aligned tilted APHs that generate a deep V-shaped groove. The conserved hydrophobic mismatch may thus be important for membrane destabilization during Tat transport, and the exact length of 12 hydrophobic residues could be a compromise between functionality and proton leakage minimization.

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Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli

Open Biology ◽

10.1098/rsob.170091 ◽

2017 ◽

Vol 7 (8) ◽

pp. 170091 ◽

Cited By ~ 11

Author(s):

Johann Habersetzer ◽

Kristoffer Moore ◽

Jon Cherry ◽

Grant Buchanan ◽

Phillip J. Stansfeld ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Protein Transport ◽

Cytoplasmic Membrane ◽

Polar Cluster ◽

Additional Sequence ◽

Tat System ◽

Folded Proteins ◽

Related Proteins

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

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Substrate-triggered position-switching of TatA and TatB is an essential step in the Escherichia coli Tat protein export pathway

10.1101/113985 ◽

2017 ◽

Cited By ~ 3

Author(s):

Johann Habersetzer ◽

Kristoffer Moore ◽

Jon Cherry ◽

Grant Buchanan ◽

Phillip Stansfeld ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Protein Transport ◽

Transmembrane Helix ◽

Tat Protein ◽

Disulfide Crosslinking ◽

Additional Sequence ◽

Tat System ◽

Folded Proteins ◽

Related Proteins

AbstractThe twin arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Here we use disulfide crosslinking and molecular modelling to show there are two binding sites for TatA/B proteins on TatC. TatA and TatB are each able to occupy both sites if they are the only TatA/B protein present. However, under resting conditions the sites are differentially occupied with TatB occupying the ‘polar cluster’ site while TatA binds adjacently at the TatC transmembrane helix 6 binding site. When the Tat system is activated by the overproduction of a substrate, TatA and TatB switch their binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

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Engineering a super-secreting strain of Escherichia coli by directed co-evolution of the multiprotein Tat translocation machinery

10.1101/2021.04.25.441317 ◽

2021 ◽

Author(s):

May N. Taw ◽

Mingji Li ◽

Daniel Kim ◽

Mark A. Rocco ◽

Dujduan Waraho-Zhmayev ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Genetic Manipulation ◽

Value Added ◽

Heterologous Proteins ◽

Sec Pathway ◽

Twin Arginine Translocation ◽

Transit Times ◽

Tat Pathway ◽

Folded Proteins

AbstractEscherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed co-evolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three super-secreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than that observed for wildtype TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of highly efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

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The Twin-Arginine Translocation System: A Novel Means of Transporting Folded Proteins in Chloroplasts and Bacteria

Biological Chemistry ◽

10.1515/bc.2000.013 ◽

2000 ◽

Vol 381 (2) ◽

pp. 89-93 ◽

Cited By ~ 14

Author(s):

C. Robinson

Keyword(s):

Escherichia Coli ◽

Transport Proteins ◽

Membrane Systems ◽

Related System ◽

Twin Arginine Translocation ◽

System A ◽

Unfolded State ◽

Tat System ◽

Folded Proteins ◽

Unique Ability

Abstract Protein translocases have been characterised in several membrane systems and the translocation mechanisms have been shown to differ in critical respects. Nevertheless, the majority were believed to transport proteins only in a largely unfolded state, and this widespread characteristic was viewed as a likely evolutionary effort to minimise the diameter of translocation pore required. Within the last few years, however, studies on the chloroplast thylakoid membrane have revealed a novel class of protein translocase which possesses the apparently unique ability to transport fullyfolded proteins across a tightly sealed energytransducing membrane. A related system, (the twinarginine translocation, or Tat system) has now been characterised in the Escherichia coli plasma membrane and considerations of its substrate specificity again point to its involvement in the transport of folded proteins. The emerging data suggest a critical involvement in many membranes for the biogenesis of two types of globular protein: those that are obliged to fold prior to translocation, and those that fold too tightly or rapidly for other types of protein translocase to handle.

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Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Microbial Biotechnology ◽

10.1111/j.1751-7915.2008.00041.x ◽

2008 ◽

Vol 1 (5) ◽

pp. 403-415 ◽

Cited By ~ 19

Author(s):

Adam C. Fisher ◽

Jae-Young Kim ◽

Ritsdeliz Perez-Rodriguez ◽

Danielle Tullman-Ercek ◽

Wallace R. Fish ◽

...

Keyword(s):

Escherichia Coli ◽

Expression And Purification ◽

Twin Arginine Translocation ◽

Folded Proteins

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Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081693 ◽

1978 ◽

Vol 36 (2) ◽

pp. 166-167 ◽

Cited By ~ 1

Author(s):

G. Stöffler ◽

R.W. Bald ◽

J. Dieckhoff ◽

H. Eckhard ◽

R. Lührmann ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Ribosomal Subunit ◽

Spatial Arrangement ◽

Small Subunit ◽

Antibody Binding ◽

Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

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Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060490 ◽

1967 ◽

Vol 25 ◽

pp. 96-97

Author(s):

Manfred E. Bayer

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Electron Microscope ◽

Uranyl Acetate ◽

E Coli ◽

Receptor Sites ◽

Rigid Cell Wall ◽

Host Cell Surface ◽

Bacterial Viruses ◽

Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

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Infection of Escherichia coli with bacteriophages

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100063767 ◽

1969 ◽

Vol 27 ◽

pp. 370-371

Author(s):

Manfred E. Bayer

Keyword(s):

Escherichia Coli ◽

Nucleic Acid ◽

Cell Surface ◽

Virus Type ◽

Infection Process ◽

Adsorption Site ◽

The Other ◽

Specific Receptors ◽

Bacterial Receptors

The first step in the infection of a bacterium by a virus consists of a collision between cell and bacteriophage. The presence of virus-specific receptors on the cell surface will trigger a number of events leading eventually to release of the phage nucleic acid. The execution of the various "steps" in the infection process varies from one virus-type to the other, depending on the anatomy of the virus. Small viruses like ØX 174 and MS2 adsorb directly with their capsid to the bacterial receptors, while other phages possess attachment organelles of varying complexity. In bacteriophages T3 (Fig. 1) and T7 the small conical processes of their heads point toward the adsorption site; a welldefined baseplate is attached to the head of P22; heads without baseplates are not infective.

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