Blue-Shifted Green Fluorescent Protein Homologues Are Brighter Than Enhanced Green Fluorescent Protein Under Two-Photon Excitation

Overview

Journal J Phys Chem Lett

Publisher American Chemical Society

Specialty Chemistry

Date 2017 May 23

PMID 28530831

Citations 20

Authors

Rosana S Molina

Tam M Tran

Robert E Campbell

Gerard G Lambert

Anya Salih

Nathan C Shaner

Thomas E Hughes

Mikhail Drobizhev

Affiliations

Soon will be listed here.

Abstract

Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, especially in the brains of small model organisms. The quantity of physiologically relevant data collected, however, is limited by heat-induced damage of the tissue due to the high intensities of the excitation laser. We seek to minimize this damage by developing FPs with improved brightness. Among FPs with the same chromophore structure, the spectral properties can vary widely due to differences in the local protein environment. Using a physical model that describes the spectra of FPs containing the anionic green FP (GFP) chromophore, we predict that those that are blue-shifted in one-photon absorption will have stronger peak two-photon absorption cross sections. Following this prediction, we present 12 blue-shifted GFP homologues and demonstrate that they are up to 2.5 times brighter than the commonly used enhanced GFP (EGFP).

Citing Articles

Blue-shifted genetically encoded Ca indicator with enhanced two-photon absorption.

Aggarwal A, Sunil S, Bendifallah I, Moon M, Drobizhev M, Zarowny L Neurophotonics. 2024; 11(2):024207.

PMID: 38577628 PMC: 10993905. DOI: 10.1117/1.NPh.11.2.024207.

A blue-shifted genetically encoded Ca indicator with enhanced two-photon absorption.

Aggarwal A, Sunil S, Bendifallah I, Moon M, Drobizhev M, Zarowny L bioRxiv. 2023; .

PMID: 37905143 PMC: 10614751. DOI: 10.1101/2023.10.12.562058.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging.

Nasu Y, Aggarwal A, Le G, Vo C, Kambe Y, Wang X Nat Commun. 2023; 14(1):6598.

PMID: 37891202 PMC: 10611801. DOI: 10.1038/s41467-023-42230-5.

Impact of the Protein Environment on Two-Photon Absorption Cross-Sections of the GFP Chromophore Anion Resolved at the XMCQDPT2 Level of Theory.

Aslopovsky V, Scherbinin A, Kleshchina N, Bochenkova A Int J Mol Sci. 2023; 24(14).

PMID: 37511026 PMC: 10379633. DOI: 10.3390/ijms241411266.

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins.

Kabir M, Ouedraogo D, Orozco-Gonzalez Y, Gadda G, Gozem S J Phys Chem B. 2023; 127(6):1301-1311.

PMID: 36740810 PMC: 9940217. DOI: 10.1021/acs.jpcb.2c06475.

References

Jacques S . Optical properties of biological tissues: a review. Phys Med Biol. 2013; 58(11):R37-61. DOI: 10.1088/0031-9155/58/11/R37. View

Drobizhev M, Callis P, Nifosi R, Wicks G, Stoltzfus C, Barnett L . Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning. Sci Rep. 2015; 5:13223. PMC: 4541067. DOI: 10.1038/srep13223. View

Patterson G, Day R, Piston D . Fluorescent protein spectra. J Cell Sci. 2001; 114(Pt 5):837-8. DOI: 10.1242/jcs.114.5.837. View

Helmchen F, Denk W . Deep tissue two-photon microscopy. Nat Methods. 2005; 2(12):932-40. DOI: 10.1038/nmeth818. View

Altoe P, Bernardi F, Garavelli M, Orlandi G, Negri F . Solvent effects on the vibrational activity and photodynamics of the green fluorescent protein chromophore: a quantum-chemical study. J Am Chem Soc. 2005; 127(11):3952-63. DOI: 10.1021/ja0451517. View

Day R, Booker C, Periasamy A . Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. J Biomed Opt. 2008; 13(3):031203. PMC: 2483694. DOI: 10.1117/1.2939094. View

Ai H, Henderson J, Remington S, Campbell R . Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem J. 2006; 400(3):531-40. PMC: 1698604. DOI: 10.1042/BJ20060874. View

Kelmanson I, Matz M . Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia: Faviida). Mol Biol Evol. 2003; 20(7):1125-33. DOI: 10.1093/molbev/msg130. View

Shaner N, Lambert G, Chammas A, Ni Y, Cranfill P, Baird M . A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods. 2013; 10(5):407-9. PMC: 3811051. DOI: 10.1038/nmeth.2413. View

10.

Zinter J, Levene M . Maximizing fluorescence collection efficiency in multiphoton microscopy. Opt Express. 2011; 19(16):15348-62. PMC: 3482884. DOI: 10.1364/OE.19.015348. View

11.

Yanushevich Y, Staroverov D, Savitsky A, Fradkov A, Gurskaya N, Bulina M . A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 2002; 511(1-3):11-4. DOI: 10.1016/s0014-5793(01)03263-x. View

12.

Sofroniew N, Flickinger D, King J, Svoboda K . A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. Elife. 2016; 5. PMC: 4951199. DOI: 10.7554/eLife.14472. View

13.

Dewey W . Arrhenius relationships from the molecule and cell to the clinic. Int J Hyperthermia. 2009; 25(1):3-20. DOI: 10.1080/02656730902747919. View

14.

Malo G, Wang M, Wu D, Stelling A, Tonge P, Wachter R . Crystal structure and Raman studies of dsFP483, a cyan fluorescent protein from Discosoma striata. J Mol Biol. 2008; 378(4):871-86. PMC: 2435309. DOI: 10.1016/j.jmb.2008.02.069. View

15.

Shaner N, Steinbach P, Tsien R . A guide to choosing fluorescent proteins. Nat Methods. 2005; 2(12):905-9. DOI: 10.1038/nmeth819. View

16.

Henderson J, Remington S . Crystal structures and mutational analysis of amFP486, a cyan fluorescent protein from Anemonia majano. Proc Natl Acad Sci U S A. 2005; 102(36):12712-7. PMC: 1200261. DOI: 10.1073/pnas.0502250102. View

17.

Filippi C, Buda F, Guidoni L, Sinicropi A . Bathochromic Shift in Green Fluorescent Protein: A Puzzle for QM/MM Approaches. J Chem Theory Comput. 2015; 8(1):112-24. DOI: 10.1021/ct200704k. View

18.

Rodriguez E, Campbell R, Lin J, Lin M, Miyawaki A, Palmer A . The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem Sci. 2016; 42(2):111-129. PMC: 5272834. DOI: 10.1016/j.tibs.2016.09.010. View

19.

KOESTER H, Baur D, Uhl R, Hell S . Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys J. 1999; 77(4):2226-36. PMC: 1300503. DOI: 10.1016/S0006-3495(99)77063-3. View

20.

Ai H, Baird M, Shen Y, Davidson M, Campbell R . Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications. Nat Protoc. 2014; 9(4):910-28. DOI: 10.1038/nprot.2014.054. View