Tim Kuekenshoener, Daniel Wohlwend, Christoph Niemoeller, Padmarupa Dondapati, Janina Speck, Adebola V. Adeniran, Anita Nieth, Stefan Gerhardt, Oliver Einsle, Kristian M. Mueller, Katja Maren Arndt
- The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of alpha-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1 angstrom)The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of alpha-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1 angstrom) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach. (C) 2014 Elsevier Inc. All rights reserved.…
MetadatenAuthor details: | Tim Kuekenshoener, Daniel Wohlwend, Christoph Niemoeller, Padmarupa Dondapati, Janina Speck, Adebola V. Adeniran, Anita Nieth, Stefan Gerhardt, Oliver Einsle, Kristian M. Mueller, Katja Maren ArndtORCiDGND |
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DOI: | https://doi.org/10.1016/j.jsb.2014.03.002 |
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ISSN: | 1047-8477 |
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ISSN: | 1095-8657 |
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Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/24631970 |
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Title of parent work (English): | Journal of structural biology |
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Publisher: | Elsevier |
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Place of publishing: | San Diego |
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Publication type: | Article |
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Language: | English |
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Year of first publication: | 2014 |
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Publication year: | 2014 |
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Release date: | 2017/03/27 |
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Tag: | Basic helix-loop-helix leucine zipper; Coiled coils; Microphthalmia associated transcription factor; Selection and design; Twin arginine translocation pathway |
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Volume: | 186 |
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Issue: | 3 |
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Number of pages: | 14 |
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First page: | 335 |
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Last Page: | 348 |
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Funding institution: | Collaborative Research Centre (SFB) 850 of the German Research
Foundation (DFG); European Research Council; Excellence Initiative of
the German Federal and State Governments [EXC 294]; DAAD-RISE program
and a Fulbright undergraduate scholarship |
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Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
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Peer review: | Referiert |
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