--- _id: '1611' 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 "core" 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. author: - first_name: Jason full_name: Whitfield, Jason last_name: Whitfield - first_name: William full_name: Zhang, William last_name: Zhang - first_name: Michel full_name: Herde, Michel last_name: Herde - first_name: Ben full_name: Clifton, Ben last_name: Clifton - first_name: Johanna full_name: Radziejewski, Johanna last_name: Radziejewski - first_name: Harald L full_name: Janovjak, Harald L id: 33BA6C30-F248-11E8-B48F-1D18A9856A87 last_name: Janovjak orcid: 0000-0002-8023-9315 - first_name: Christian full_name: Henneberger, Christian last_name: Henneberger - first_name: Colin full_name: Jackson, Colin last_name: Jackson citation: ama: Whitfield J, Zhang W, Herde M, et al. Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction. Protein Science. 2015;24(9):1412-1422. doi:10.1002/pro.2721 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. Protein Science. Wiley. https://doi.org/10.1002/pro.2721 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.” Protein Science. Wiley, 2015. https://doi.org/10.1002/pro.2721. ieee: J. Whitfield et al., “Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction,” Protein Science, 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. mla: Whitfield, Jason, et al. “Construction of a Robust and Sensitive Arginine Biosensor through Ancestral Protein Reconstruction.” Protein Science, vol. 24, no. 9, Wiley, 2015, pp. 1412–22, doi:10.1002/pro.2721. short: J. Whitfield, W. Zhang, M. Herde, B. Clifton, J. Radziejewski, H.L. Janovjak, C. Henneberger, C. Jackson, Protein Science 24 (2015) 1412–1422. date_created: 2018-12-11T11:53:01Z date_published: 2015-09-01T00:00:00Z date_updated: 2021-01-12T06:52:00Z day: '01' department: - _id: HaJa doi: 10.1002/pro.2721 external_id: pmid: - '26061224' intvolume: ' 24' issue: '9' language: - iso: eng main_file_link: - open_access: '1' url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570536/ month: '09' oa: 1 oa_version: Submitted Version page: 1412 - 1422 pmid: 1 project: - _id: 255BFFFA-B435-11E9-9278-68D0E5697425 grant_number: RGY0084/2012 name: In situ real-time imaging of neurotransmitter signaling using designer optical sensors (HFSP Young Investigator) publication: Protein Science publication_status: published publisher: Wiley publist_id: '5555' quality_controlled: '1' scopus_import: 1 status: public title: Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 24 year: '2015' ...