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. 2022 Mar 7;12:3678. doi: 10.1038/s41598-022-07313-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Engineering and characterization of mCyRFP3. Engineering of mCyRFP3. (a) Crystal structure of dimeric predecessor CyOFP1 displaying interacting residues Y148 and A161. Image was generated in Schrödinger MacPyMol 1.8. (b) Size-exclusion chromatography of EGFP, tdTomato, CyRFP1, and mCyRFP3. EGFP was used as a monomeric standard, while tdTomato was used as a dimeric standard. (c) Excitation and emission spectra of mCyRFP3 compared to its parent mCyRFP1. (d) pH dependence of mCyRFP3 fluorescence, demonstrating a pKa of 4.1. Error bars are s.em.m of triplicate measurements. (e) Photobleaching kinetics of purified cyan- excitable red fluorescent proteins under illumination by a 120-W metal-halide arc lamp through a 490/20-nm excitation filter. The time-axis was adjusted for each fluorophore to simulate excitation conditions producing 1000 photons per s per molecule. Lighter shading on CyRFP1, mCyRFP1, and mCyRFP3 lines represents standard deviation of five measurements. (f) Maturation kinetics of mCyRFP3 demonstrating a half-life of t = 12.5 min. Graphs in (a) through (f) were generated in Microsoft Excel for Mac 16.