[{"date_published":"2017-03-15T00:00:00Z","_id":"957","author":[{"full_name":"Clifton, Ben","first_name":"Ben","last_name":"Clifton"},{"full_name":"Whitfield, Jason","first_name":"Jason","last_name":"Whitfield"},{"full_name":"Sanchez Romero, Inmaculada","first_name":"Inmaculada","last_name":"Sanchez Romero","id":"3D9C5D30-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Herde","first_name":"Michel","full_name":"Herde, Michel"},{"full_name":"Henneberger, Christian","first_name":"Christian","last_name":"Henneberger"},{"first_name":"Harald L","full_name":"Janovjak, Harald L","orcid":"0000-0002-8023-9315","last_name":"Janovjak","id":"33BA6C30-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Jackson","first_name":"Colin","full_name":"Jackson, Colin"}],"type":"book_chapter","year":"2017","publisher":"Springer","doi":"10.1007/978-1-4939-6940-1_5","language":[{"iso":"eng"}],"volume":1596,"publist_id":"6451","publication_identifier":{"issn":["10643745"]},"quality_controlled":"1","month":"03","intvolume":"      1596","user_id":"4435EBFC-F248-11E8-B48F-1D18A9856A87","department":[{"_id":"HaJa"}],"date_created":"2018-12-11T11:49:24Z","abstract":[{"text":"Small molecule biosensors based on Forster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.","lang":"eng"}],"day":"15","page":"71 - 87","publication_status":"published","oa_version":"None","status":"public","date_updated":"2021-01-12T08:22:13Z","title":"Ancestral protein reconstruction and circular permutation for improving the stability and dynamic range of FRET sensors","series_title":"Synthetic Protein Switches","publication":"Synthetic Protein Switches","alternative_title":["Methods in Molecular Biology"],"editor":[{"first_name":"Viktor","full_name":"Stein, Viktor","last_name":"Stein"}],"scopus_import":1,"citation":{"short":"B. Clifton, J. Whitfield, I. Sanchez-Romero, M. Herde, C. Henneberger, H.L. Janovjak, C. Jackson, in:, V. Stein (Ed.), Synthetic Protein Switches, Springer, 2017, pp. 71–87.","mla":"Clifton, Ben, et al. “Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors.” <i>Synthetic Protein Switches</i>, edited by Viktor Stein, vol. 1596, Springer, 2017, pp. 71–87, doi:<a href=\"https://doi.org/10.1007/978-1-4939-6940-1_5\">10.1007/978-1-4939-6940-1_5</a>.","ista":"Clifton B, Whitfield J, Sanchez-Romero I, Herde M, Henneberger C, Janovjak HL, Jackson C. 2017.Ancestral protein reconstruction and circular permutation for improving the stability and dynamic range of FRET sensors. In: Synthetic Protein Switches. Methods in Molecular Biology, vol. 1596, 71–87.","apa":"Clifton, B., Whitfield, J., Sanchez-Romero, I., Herde, M., Henneberger, C., Janovjak, H. L., &#38; Jackson, C. (2017). Ancestral protein reconstruction and circular permutation for improving the stability and dynamic range of FRET sensors. In V. Stein (Ed.), <i>Synthetic Protein Switches</i> (Vol. 1596, pp. 71–87). Springer. <a href=\"https://doi.org/10.1007/978-1-4939-6940-1_5\">https://doi.org/10.1007/978-1-4939-6940-1_5</a>","ama":"Clifton B, Whitfield J, Sanchez-Romero I, et al. Ancestral protein reconstruction and circular permutation for improving the stability and dynamic range of FRET sensors. In: Stein V, ed. <i>Synthetic Protein Switches</i>. Vol 1596. Synthetic Protein Switches. Springer; 2017:71-87. doi:<a href=\"https://doi.org/10.1007/978-1-4939-6940-1_5\">10.1007/978-1-4939-6940-1_5</a>","chicago":"Clifton, Ben, Jason Whitfield, Inmaculada Sanchez-Romero, Michel Herde, Christian Henneberger, Harald L Janovjak, and Colin Jackson. “Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors.” In <i>Synthetic Protein Switches</i>, edited by Viktor Stein, 1596:71–87. Synthetic Protein Switches. Springer, 2017. <a href=\"https://doi.org/10.1007/978-1-4939-6940-1_5\">https://doi.org/10.1007/978-1-4939-6940-1_5</a>.","ieee":"B. Clifton <i>et al.</i>, “Ancestral protein reconstruction and circular permutation for improving the stability and dynamic range of FRET sensors,” in <i>Synthetic Protein Switches</i>, vol. 1596, V. Stein, Ed. Springer, 2017, pp. 71–87."},"project":[{"grant_number":"RGY0084/2012","_id":"255BFFFA-B435-11E9-9278-68D0E5697425","name":"In situ real-time imaging of neurotransmitter signaling using designer optical sensors (HFSP Young Investigator)"}]},{"_id":"958","author":[{"last_name":"Mitchell","full_name":"Mitchell, Joshua","first_name":"Joshua"},{"first_name":"William","full_name":"Zhang, William","last_name":"Zhang"},{"full_name":"Herde, Michel","first_name":"Michel","last_name":"Herde"},{"first_name":"Christian","full_name":"Henneberger, Christian","last_name":"Henneberger"},{"full_name":"Janovjak, Harald L","first_name":"Harald L","orcid":"0000-0002-8023-9315","id":"33BA6C30-F248-11E8-B48F-1D18A9856A87","last_name":"Janovjak"},{"first_name":"Megan","full_name":"O'Mara, Megan","last_name":"O'Mara"},{"first_name":"Colin","full_name":"Jackson, Colin","last_name":"Jackson"}],"date_published":"2017-05-15T00:00:00Z","publisher":"Springer","year":"2017","language":[{"iso":"eng"}],"doi":"10.1007/978-1-4939-6940-1_6","type":"book_chapter","quality_controlled":"1","publist_id":"6450","publication_identifier":{"issn":["10643745"]},"month":"05","volume":1596,"department":[{"_id":"HaJa"}],"date_created":"2018-12-11T11:49:24Z","intvolume":"      1596","user_id":"4435EBFC-F248-11E8-B48F-1D18A9856A87","abstract":[{"lang":"eng","text":"Biosensors that exploit Forster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors."}],"page":"89 - 99","publication_status":"published","oa_version":"None","day":"15","date_updated":"2021-01-12T08:22:13Z","status":"public","title":"Method for developing optical sensors using a synthetic dye fluorescent protein FRET pair and computational modeling and assessment","publication":"Synthetic Protein Switches","series_title":"Synthetic Protein Switches","citation":{"ieee":"J. Mitchell <i>et al.</i>, “Method for developing optical sensors using a synthetic dye fluorescent protein FRET pair and computational modeling and assessment,” in <i>Synthetic Protein Switches</i>, vol. 1596, V. Stein, Ed. Springer, 2017, pp. 89–99.","chicago":"Mitchell, Joshua, William Zhang, Michel Herde, Christian Henneberger, Harald L Janovjak, Megan O’Mara, and Colin Jackson. “Method for Developing Optical Sensors Using a Synthetic Dye Fluorescent Protein FRET Pair and Computational Modeling and Assessment.” In <i>Synthetic Protein Switches</i>, edited by Viktor Stein, 1596:89–99. Synthetic Protein Switches. Springer, 2017. <a href=\"https://doi.org/10.1007/978-1-4939-6940-1_6\">https://doi.org/10.1007/978-1-4939-6940-1_6</a>.","ama":"Mitchell J, Zhang W, Herde M, et al. Method for developing optical sensors using a synthetic dye fluorescent protein FRET pair and computational modeling and assessment. In: Stein V, ed. <i>Synthetic Protein Switches</i>. Vol 1596. Synthetic Protein Switches. Springer; 2017:89-99. doi:<a href=\"https://doi.org/10.1007/978-1-4939-6940-1_6\">10.1007/978-1-4939-6940-1_6</a>","short":"J. Mitchell, W. Zhang, M. Herde, C. Henneberger, H.L. Janovjak, M. O’Mara, C. Jackson, in:, V. Stein (Ed.), Synthetic Protein Switches, Springer, 2017, pp. 89–99.","mla":"Mitchell, Joshua, et al. “Method for Developing Optical Sensors Using a Synthetic Dye Fluorescent Protein FRET Pair and Computational Modeling and Assessment.” <i>Synthetic Protein Switches</i>, edited by Viktor Stein, vol. 1596, Springer, 2017, pp. 89–99, doi:<a href=\"https://doi.org/10.1007/978-1-4939-6940-1_6\">10.1007/978-1-4939-6940-1_6</a>.","apa":"Mitchell, J., Zhang, W., Herde, M., Henneberger, C., Janovjak, H. L., O’Mara, M., &#38; Jackson, C. (2017). Method for developing optical sensors using a synthetic dye fluorescent protein FRET pair and computational modeling and assessment. In V. Stein (Ed.), <i>Synthetic Protein Switches</i> (Vol. 1596, pp. 89–99). Springer. <a href=\"https://doi.org/10.1007/978-1-4939-6940-1_6\">https://doi.org/10.1007/978-1-4939-6940-1_6</a>","ista":"Mitchell J, Zhang W, Herde M, Henneberger C, Janovjak HL, O’Mara M, Jackson C. 2017.Method for developing optical sensors using a synthetic dye fluorescent protein FRET pair and computational modeling and assessment. In: Synthetic Protein Switches. Methods in Molecular Biology, vol. 1596, 89–99."},"alternative_title":["Methods in Molecular Biology"],"editor":[{"full_name":"Stein, Viktor","first_name":"Viktor","last_name":"Stein"}],"scopus_import":1},{"publication":"Auxins and Cytokinins in Plant Biology","related_material":{"record":[{"id":"539","status":"public","relation":"dissertation_contains"}]},"file":[{"date_updated":"2019-10-15T07:47:05Z","file_id":"5068","date_created":"2018-12-12T10:14:18Z","file_size":840646,"file_name":"IST-2018-1019-v1+1_Hurny_MethodsMolBiol_2017.pdf","relation":"main_file","content_type":"application/pdf","access_level":"open_access","creator":"system"}],"title":"Methodological advances in auxin and cytokinin biology","scopus_import":1,"alternative_title":["Methods in Molecular Biology"],"has_accepted_license":"1","pubrep_id":"1019","project":[{"name":"Hormone cross-talk drives nutrient dependent plant development","_id":"2542D156-B435-11E9-9278-68D0E5697425","grant_number":"I 1774-B16","call_identifier":"FWF"}],"citation":{"ieee":"A. Hurny and E. Benková, “Methodological advances in auxin and cytokinin biology,” <i>Auxins and Cytokinins in Plant Biology</i>, vol. 1569. Springer, pp. 1–29, 2017.","chicago":"Hurny, Andrej, and Eva Benková. “Methodological Advances in Auxin and Cytokinin Biology.” <i>Auxins and Cytokinins in Plant Biology</i>. Springer, 2017. <a href=\"https://doi.org/10.1007/978-1-4939-6831-2_1\">https://doi.org/10.1007/978-1-4939-6831-2_1</a>.","ama":"Hurny A, Benková E. Methodological advances in auxin and cytokinin biology. <i>Auxins and Cytokinins in Plant Biology</i>. 2017;1569:1-29. doi:<a href=\"https://doi.org/10.1007/978-1-4939-6831-2_1\">10.1007/978-1-4939-6831-2_1</a>","ista":"Hurny A, Benková E. 2017. Methodological advances in auxin and cytokinin biology. Auxins and Cytokinins in Plant Biology. 1569, 1–29.","mla":"Hurny, Andrej, and Eva Benková. “Methodological Advances in Auxin and Cytokinin Biology.” <i>Auxins and Cytokinins in Plant Biology</i>, vol. 1569, Springer, 2017, pp. 1–29, doi:<a href=\"https://doi.org/10.1007/978-1-4939-6831-2_1\">10.1007/978-1-4939-6831-2_1</a>.","short":"A. Hurny, E. Benková, Auxins and Cytokinins in Plant Biology 1569 (2017) 1–29.","apa":"Hurny, A., &#38; Benková, E. (2017). Methodological advances in auxin and cytokinin biology. <i>Auxins and Cytokinins in Plant Biology</i>. Springer. <a href=\"https://doi.org/10.1007/978-1-4939-6831-2_1\">https://doi.org/10.1007/978-1-4939-6831-2_1</a>"},"oa_version":"Submitted Version","publication_status":"published","page":"1 - 29","day":"17","abstract":[{"text":"The history of auxin and cytokinin biology including the initial discoveries by father–son duo Charles Darwin and Francis Darwin (1880), and Gottlieb Haberlandt (1919) is a beautiful demonstration of unceasing continuity of research. Novel findings are integrated into existing hypotheses and models and deepen our understanding of biological principles. At the same time new questions are triggered and hand to hand with this new methodologies are developed to address these new challenges.","lang":"eng"}],"status":"public","date_updated":"2024-03-25T23:30:09Z","volume":1569,"month":"03","quality_controlled":"1","publist_id":"6369","publication_identifier":{"issn":["10643745"]},"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","ddc":["575"],"file_date_updated":"2019-10-15T07:47:05Z","intvolume":"      1569","date_created":"2018-12-11T11:49:45Z","department":[{"_id":"EvBe"}],"date_published":"2017-03-17T00:00:00Z","author":[{"full_name":"Hurny, Andrej","first_name":"Andrej","orcid":"0000-0003-3638-1426","id":"4DC4AF46-F248-11E8-B48F-1D18A9856A87","last_name":"Hurny"},{"full_name":"Benková, Eva","first_name":"Eva","orcid":"0000-0002-8510-9739","id":"38F4F166-F248-11E8-B48F-1D18A9856A87","last_name":"Benková"}],"oa":1,"_id":"1024","type":"journal_article","language":[{"iso":"eng"}],"doi":"10.1007/978-1-4939-6831-2_1","publisher":"Springer","year":"2017"},{"abstract":[{"text":"Exogenous application of biologically important molecules for plant growth promotion and/or regulation is very common both in plant research and horticulture. Plant hormones such as auxins and cytokinins are classes of compounds which are often applied exogenously. Nevertheless, plants possess a well-established machinery to regulate the active pool of exogenously applied compounds by converting them to metabolites and conjugates. Consequently, it is often very useful to know the in vivo status of applied compounds to connect them with some of the regulatory events in plant developmental processes. The in vivo status of applied compounds can be measured by incubating plants with radiolabeled compounds, followed by extraction, purification, and HPLC metabolic profiling of plant extracts. Recently we have used this method to characterize the intracellularly localized PIN protein, PIN5. Here we explain the method in detail, with a focus on general application. ","lang":"eng"}],"day":"01","page":"255 - 264","oa_version":"None","publication_status":"published","status":"public","date_updated":"2021-01-12T06:56:15Z","title":"Analyzing the in vivo status of exogenously applied auxins: A HPLC-based method to characterize the intracellularly localized auxin transporters","series_title":"Methods in Molecular Biology","publication":"Plant Chemical Genomics","alternative_title":["Methods in Molecular Biology"],"editor":[{"first_name":"Glenn","full_name":"Hicks, Glenn","last_name":"Hicks"},{"last_name":"Robert","first_name":"Stéphanie","full_name":"Robert, Stéphanie"}],"scopus_import":1,"citation":{"ama":"Simon S, Skůpa P, Dobrev P, Petrášek J, Zažímalová E, Friml J. Analyzing the in vivo status of exogenously applied auxins: A HPLC-based method to characterize the intracellularly localized auxin transporters. In: Hicks G, Robert S, eds. <i>Plant Chemical Genomics</i>. Vol 1056. Methods in Molecular Biology. Springer; 2014:255-264. doi:<a href=\"https://doi.org/10.1007/978-1-62703-592-7_23\">10.1007/978-1-62703-592-7_23</a>","ista":"Simon S, Skůpa P, Dobrev P, Petrášek J, Zažímalová E, Friml J. 2014.Analyzing the in vivo status of exogenously applied auxins: A HPLC-based method to characterize the intracellularly localized auxin transporters. In: Plant Chemical Genomics. Methods in Molecular Biology, vol. 1056, 255–264.","mla":"Simon, Sibu, et al. “Analyzing the in Vivo Status of Exogenously Applied Auxins: A HPLC-Based Method to Characterize the Intracellularly Localized Auxin Transporters.” <i>Plant Chemical Genomics</i>, edited by Glenn Hicks and Stéphanie Robert, vol. 1056, Springer, 2014, pp. 255–64, doi:<a href=\"https://doi.org/10.1007/978-1-62703-592-7_23\">10.1007/978-1-62703-592-7_23</a>.","short":"S. Simon, P. Skůpa, P. Dobrev, J. Petrášek, E. Zažímalová, J. Friml, in:, G. Hicks, S. Robert (Eds.), Plant Chemical Genomics, Springer, 2014, pp. 255–264.","apa":"Simon, S., Skůpa, P., Dobrev, P., Petrášek, J., Zažímalová, E., &#38; Friml, J. (2014). Analyzing the in vivo status of exogenously applied auxins: A HPLC-based method to characterize the intracellularly localized auxin transporters. In G. Hicks &#38; S. Robert (Eds.), <i>Plant Chemical Genomics</i> (Vol. 1056, pp. 255–264). Springer. <a href=\"https://doi.org/10.1007/978-1-62703-592-7_23\">https://doi.org/10.1007/978-1-62703-592-7_23</a>","ieee":"S. Simon, P. Skůpa, P. Dobrev, J. Petrášek, E. Zažímalová, and J. Friml, “Analyzing the in vivo status of exogenously applied auxins: A HPLC-based method to characterize the intracellularly localized auxin transporters,” in <i>Plant Chemical Genomics</i>, vol. 1056, G. Hicks and S. Robert, Eds. Springer, 2014, pp. 255–264.","chicago":"Simon, Sibu, Petr Skůpa, Petre Dobrev, Jan Petrášek, Eva Zažímalová, and Jiří Friml. “Analyzing the in Vivo Status of Exogenously Applied Auxins: A HPLC-Based Method to Characterize the Intracellularly Localized Auxin Transporters.” In <i>Plant Chemical Genomics</i>, edited by Glenn Hicks and Stéphanie Robert, 1056:255–64. Methods in Molecular Biology. Springer, 2014. <a href=\"https://doi.org/10.1007/978-1-62703-592-7_23\">https://doi.org/10.1007/978-1-62703-592-7_23</a>."},"date_published":"2014-01-01T00:00:00Z","_id":"2245","author":[{"full_name":"Simon, Sibu","first_name":"Sibu","orcid":"0000-0002-1998-6741","id":"4542EF9A-F248-11E8-B48F-1D18A9856A87","last_name":"Simon"},{"first_name":"Petr","full_name":"Skůpa, Petr","last_name":"Skůpa"},{"last_name":"Dobrev","first_name":"Petre","full_name":"Dobrev, Petre"},{"first_name":"Jan","full_name":"Petrášek, Jan","last_name":"Petrášek"},{"first_name":"Eva","full_name":"Zažímalová, Eva","last_name":"Zažímalová"},{"full_name":"Friml, Jirí","first_name":"Jirí","orcid":"0000-0002-8302-7596","id":"4159519E-F248-11E8-B48F-1D18A9856A87","last_name":"Friml"}],"type":"book_chapter","year":"2014","publisher":"Springer","doi":"10.1007/978-1-62703-592-7_23","language":[{"iso":"eng"}],"volume":1056,"publist_id":"4704","publication_identifier":{"issn":["10643745"]},"quality_controlled":"1","month":"01","intvolume":"      1056","user_id":"4435EBFC-F248-11E8-B48F-1D18A9856A87","department":[{"_id":"JiFr"}],"date_created":"2018-12-11T11:56:32Z"}]