Design of the linkers which effectively separate domains of a bifunctional fusion protein

Arai, Ryoichi; Ueda, Hiroshi; Kitayama, Atsushi; Kamiya, Noriho; Nagamune, Teruyuki

doi:10.1093/protein/14.8.529

Cited by 568 publications

(434 citation statements)

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“…The TatA and YFP domains of the fusion are separated by a rigid linker sequence (32), and the YFP domain contains the dimerization-suppressing A206K substitution (33). The resulting strains are referred to according to the Tat proteins they produce, with "Ay" being the TatA-YFP fusion (Table S1).…”

Section: Coexpression With Tate Improves the Transport Activity Of Cellsmentioning

confidence: 99%

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Alcock

Baker

Greene

et al. 2013

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

170

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The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

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Section: Coexpression With Tate Improves the Transport Activity Of Cellsmentioning

confidence: 99%

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Alcock

Baker

Greene

et al. 2013

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

170

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“…32 Recombinant chimeric fusion proteins are routinely constructed to increase the expression of soluble proteins and to facilitate protein purification. 32,83 Other engineering approaches that link two proteins or protein domains by a peptide linker include immunoassays (e.g., using chimeras between antibody fragments and proteins 84,85 ), selection and production of antibodies, 86 and engineering of bifunctional enzymes. 87 In the respiratory chain, electron transfer protein domains of flavodoxin and cytochrome c553 from Desulfovibrio vulgaris and the heme domain of P450 BM3 from Bacillus megaterium have been used as molecular ''Lego''-type building blocks in different combinations to build artificial redox chains having variable redox potentials.…”

Section: Design Of Chimeric Proteins With Engineered Domains and Linkersmentioning

confidence: 99%

Control of protein functional dynamics by peptide linkers

2005

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Control of structural flexibility is essential for the proper functioning of a large number of proteins and multiprotein complexes. At the residue level, such flexibility occurs due to local relaxation of peptide bond angles whose cumulative effect may result in large changes in the secondary, tertiary or quaternary structures of protein molecules. Such flexibility, and its absence, most often depends on the nature of interdomain linkages formed by oligopeptides. Both flexible and relatively rigid peptide linkers are found in many multidomain proteins. Linkers are thought to control favorable and unfavorable interactions between adjacent domains by means of variable softness furnished by their primary sequence. Large-scale structural heterogeneity of multidomain proteins and their complexes, facilitated by soft peptide linkers, is now seen as the norm rather than the exception. Biophysical discoveries as well as computational algorithms and databases have reshaped our understanding of the often spectacular biomolecular dynamics enabled by soft linkers. Absence of such motion, as in so-called molecular rulers, also has desirable functional effects in protein architecture. We review here the historic discovery and current understanding of the nature of domains and their linkers from a structural, computational, and biophysical point of view. A number of emerging applications, based on the current understanding of the structural properties

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“…Because GFP does not normally fluoresce in the periplasm (23), an N-terminal GFP-TonB fusion was constructed; the fusion design was based on a previous study and included a short helical linker to enhance expression ( Fig. 2A) (22,24). The anhydrotetracycline (aTc)-inducible P LtetO-1 promoter (25) regulated crRNA and taRNA expression for the first series of experiments described below.…”

Section: Resultsmentioning

confidence: 99%

Tracking, tuning, and terminating microbial physiology using synthetic riboregulators

Callura

Dwyer

Isaacs

et al. 2010

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

166

162

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The development of biomolecular devices that interface with biological systems to reveal new insights and produce novel functions is one of the defining goals of synthetic biology. Our lab previously described a synthetic, riboregulator system that affords for modular, tunable, and tight control of gene expression in vivo. Here we highlight several experimental advantages unique to this RNA-based system, including physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and independent regulation of multiple genes. We demonstrate this utility in four sets of in vivo experiments with various microbial systems. Specifically, we show that the synthetic riboregulator is well suited for GFP fusion protein tracking in wildtype cells, tight regulation of toxic protein expression, and sensitive perturbation of stress response networks. We also show that the system can be used for logic-based computing of multiple, orthogonal inputs, resulting in the development of a programmable kill switch for bacteria. This work establishes a broad, easy-to-use synthetic biology platform for microbiology experiments and biotechnology applications. S ynthetic biology seeks to reprogram organisms to alter natural biological behavior or perform novel functions, using engineered gene circuits, pathways, and whole genomes (1-8). Engineered gene circuits, in this regard, are typically constructed by assembling well-characterized biological components into unique architectures to achieve these unnatural capabilities, which are commonly used in biotechnology applications. A need also exists for synthetic biomolecular devices that interface with endogenous systems to track, probe, and influence, rather than reprogram, cellular physiology. Synthetic gene expression platforms that could accomplish this task would be well suited for use in a wide variety of phenotypic and genetic analyses aimed at expanding our knowledge of natural biomolecular components and networks, as well as enhancing our understanding of network dynamics under an array of conditions.In this work, we showcase an engineered riboregulator system that controls gene expression posttranscriptionally through highly specific RNA-RNA interactions (9). RNA molecules are useful components in synthetic biomolecular devices (10, 11) due to their large sequence space, the number of unique structures that can be adopted, the predictability of structure stability, and the range of functions enabled by these structures. Together, these features make RNA molecules ideal for interfacing with biological systems given that they can be used to control gene expression, sense biomolecules, and serve as foundational devices that can perform complex cellular behavior. As such, the engineered, RNA-based device space is remarkably diverse. Other successful examples include independent transcription-translation networks based on orthogonal mRNA-ribosome pairs (12), riboswitches in which RNA aptamers directly control translati...

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Design of the linkers which effectively separate domains of a bifunctional fusion protein

Cited by 568 publications

References 0 publications

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Control of protein functional dynamics by peptide linkers

Tracking, tuning, and terminating microbial physiology using synthetic riboregulators

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