{"date_created":"2018-12-11T11:53:01Z","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa_version":"Submitted Version","oa":1,"scopus_import":1,"publisher":"Wiley","status":"public","title":"Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction","quality_controlled":"1","day":"01","type":"journal_article","date_updated":"2021-01-12T06:52:00Z","pmid":1,"month":"09","issue":"9","date_published":"2015-09-01T00:00:00Z","publication_status":"published","abstract":[{"lang":"eng","text":"Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding &quot;core&quot; that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 μM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue."}],"main_file_link":[{"open_access":"1","url":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570536/"}],"department":[{"_id":"HaJa"}],"volume":24,"publist_id":"5555","intvolume":"        24","year":"2015","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)"}],"publication":"Protein Science","author":[{"last_name":"Whitfield","first_name":"Jason","full_name":"Whitfield, Jason"},{"last_name":"Zhang","first_name":"William","full_name":"Zhang, William"},{"full_name":"Herde, Michel","first_name":"Michel","last_name":"Herde"},{"last_name":"Clifton","first_name":"Ben","full_name":"Clifton, Ben"},{"full_name":"Radziejewski, Johanna","first_name":"Johanna","last_name":"Radziejewski"},{"id":"33BA6C30-F248-11E8-B48F-1D18A9856A87","full_name":"Janovjak, Harald L","first_name":"Harald L","last_name":"Janovjak","orcid":"0000-0002-8023-9315"},{"first_name":"Christian","last_name":"Henneberger","full_name":"Henneberger, Christian"},{"first_name":"Colin","last_name":"Jackson","full_name":"Jackson, Colin"}],"doi":"10.1002/pro.2721","page":"1412 - 1422","external_id":{"pmid":["26061224"]},"citation":{"mla":"Whitfield, Jason, et al. “Construction of a Robust and Sensitive Arginine Biosensor through Ancestral Protein Reconstruction.” <i>Protein Science</i>, vol. 24, no. 9, Wiley, 2015, pp. 1412–22, doi:<a href=\"https://doi.org/10.1002/pro.2721\">10.1002/pro.2721</a>.","ieee":"J. Whitfield <i>et al.</i>, “Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction,” <i>Protein Science</i>, vol. 24, no. 9. Wiley, pp. 1412–1422, 2015.","ista":"Whitfield J, Zhang W, Herde M, Clifton B, Radziejewski J, Janovjak HL, Henneberger C, Jackson C. 2015. Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction. Protein Science. 24(9), 1412–1422.","chicago":"Whitfield, Jason, William Zhang, Michel Herde, Ben Clifton, Johanna Radziejewski, Harald L Janovjak, Christian Henneberger, and Colin Jackson. “Construction of a Robust and Sensitive Arginine Biosensor through Ancestral Protein Reconstruction.” <i>Protein Science</i>. Wiley, 2015. <a href=\"https://doi.org/10.1002/pro.2721\">https://doi.org/10.1002/pro.2721</a>.","apa":"Whitfield, J., Zhang, W., Herde, M., Clifton, B., Radziejewski, J., Janovjak, H. L., … Jackson, C. (2015). Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction. <i>Protein Science</i>. Wiley. <a href=\"https://doi.org/10.1002/pro.2721\">https://doi.org/10.1002/pro.2721</a>","ama":"Whitfield J, Zhang W, Herde M, et al. Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction. <i>Protein Science</i>. 2015;24(9):1412-1422. doi:<a href=\"https://doi.org/10.1002/pro.2721\">10.1002/pro.2721</a>","short":"J. Whitfield, W. Zhang, M. Herde, B. Clifton, J. Radziejewski, H.L. Janovjak, C. Henneberger, C. Jackson, Protein Science 24 (2015) 1412–1422."},"_id":"1611","language":[{"iso":"eng"}]}