Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein

Overview

Journal Biosensors (Basel)

Specialty Biotechnology

Date 2024 Jan 26

PMID 38275307

Authors

Hao Zhang

Xiaodong Tian

Jing Zhang

Hui-Wang Ai

Affiliations

Soon will be listed here.

Abstract

Introducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In this study, we optimized expression conditions for an aY-derived cpGFP (aY-cpGFP). Key factors like rich culture media and oxygen restriction pre- and post-induction enabled high-yield, high-purity production of the red-shifted protein. We also engineered two variants of aY-cpGFP with enhanced brightness by mutating a few amino acid residues surrounding the chromophore. We further investigated the sensitivity of the aY-derived protein to metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Incorporating aY into cpGFP had minimal impact on metal ion reactivity but increased the response to RNS. Expanding on these findings, we examined aY-cpGFP expression in mammalian cells and found that reductants in the culture media significantly increased the red-emitting product. Our study indicates that optimizing expression conditions to promote a reduced cellular state proved effective in producing the desired red-emitting product in both and mammalian cells, while targeted mutagenesis-based protein engineering can further enhance brightness and increase method robustness.

References

Tsien R . Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture). Angew Chem Int Ed Engl. 2009; 48(31):5612-26. DOI: 10.1002/anie.200901916. View

Nasu Y, Shen Y, Kramer L, Campbell R . Structure- and mechanism-guided design of single fluorescent protein-based biosensors. Nat Chem Biol. 2021; 17(5):509-518. DOI: 10.1038/s41589-020-00718-x. View

Mishin A, Subach F, Yampolsky I, King W, Lukyanov K, Verkhusha V . The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore. Biochemistry. 2008; 47(16):4666-73. PMC: 2900791. DOI: 10.1021/bi702130s. View

Moeyaert B, Nguyen Bich N, De Zitter E, Rocha S, Clays K, Mizuno H . Green-to-red photoconvertible Dronpa mutant for multimodal super-resolution fluorescence microscopy. ACS Nano. 2014; 8(2):1664-73. DOI: 10.1021/nn4060144. View

Baird G, Zacharias D, Tsien R . Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci U S A. 1999; 96(20):11241-6. PMC: 18018. DOI: 10.1073/pnas.96.20.11241. View

Gurskaya N, Verkhusha V, Shcheglov A, Staroverov D, Chepurnykh T, Fradkov A . Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol. 2006; 24(4):461-5. DOI: 10.1038/nbt1191. View

Imamura H, Otsubo S, Nishida M, Takekawa N, Imada K . Red fluorescent proteins engineered from green fluorescent proteins. Proc Natl Acad Sci U S A. 2023; 120(45):e2307687120. PMC: 10636333. DOI: 10.1073/pnas.2307687120. View

Gadella Jr T, van Weeren L, Stouthamer J, Hink M, Wolters A, Giepmans B . mScarlet3: a brilliant and fast-maturing red fluorescent protein. Nat Methods. 2023; 20(4):541-545. DOI: 10.1038/s41592-023-01809-y. View

Shaner N, Campbell R, Steinbach P, Giepmans B, Palmer A, Tsien R . Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004; 22(12):1567-72. DOI: 10.1038/nbt1037. View

10.

Saha R, Verma P, Rakshit S, Saha S, Mayor S, Kumar Pal S . Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins. Sci Rep. 2013; 3:1580. PMC: 3615570. DOI: 10.1038/srep01580. View

11.

Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A . An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A. 2002; 99(20):12651-6. PMC: 130515. DOI: 10.1073/pnas.202320599. View

12.

Topell S, Hennecke J, Glockshuber R . Circularly permuted variants of the green fluorescent protein. FEBS Lett. 1999; 457(2):283-9. DOI: 10.1016/s0014-5793(99)01044-3. View

13.

Wang L, Xie J, Deniz A, Schultz P . Unnatural amino acid mutagenesis of green fluorescent protein. J Org Chem. 2003; 68(1):174-6. DOI: 10.1021/jo026570u. View

14.

Elowitz M, Surette M, Wolf P, Stock J, Leibler S . Photoactivation turns green fluorescent protein red. Curr Biol. 1997; 7(10):809-12. DOI: 10.1016/s0960-9822(06)00342-3. View

15.

Corker H, Poole R . Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase, the NO-sensing regulator Fnr, and flavohemoglobin Hmp. J Biol Chem. 2003; 278(34):31584-92. DOI: 10.1074/jbc.M303282200. View

16.

Bogdanov A, Mishin A, Yampolsky I, Belousov V, Chudakov D, Subach F . Green fluorescent proteins are light-induced electron donors. Nat Chem Biol. 2009; 5(7):459-61. PMC: 2784199. DOI: 10.1038/nchembio.174. View

17.

Sawin K, Nurse P . Photoactivation of green fluorescent protein. Curr Biol. 1997; 7(10):R606-7. DOI: 10.1016/s0960-9822(06)00313-7. View

18.

Pedelacq J, Cabantous S, Tran T, Terwilliger T, Waldo G . Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2005; 24(1):79-88. DOI: 10.1038/nbt1172. View

19.

Wu T, Kumar M, Zhang J, Zhao S, Drobizhev M, McCollum M . A genetically encoded far-red fluorescent indicator for imaging synaptically released Zn. Sci Adv. 2023; 9(9):eadd2058. PMC: 9977179. DOI: 10.1126/sciadv.add2058. View

20.

Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A . EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A. 2004; 101(45):15905-10. PMC: 528746. DOI: 10.1073/pnas.0403668101. View