Fluorescent molecules are like antennas: The rate at which they absorb light depends on their orientation with respect to the incoming light wave, and the apparent intensity of their emission depends on their orientation with respect to the observer. However, the directions along which the most important fluorescent molecules in biology, fluorescent proteins (FPs), absorb and emit light are generally not known. Our optical and X-ray investigations of FP crystals have now allowed us to determine the molecular orientations of the excitation and emission transition dipole moments in the FPs mTurquoise2, eGFP, and mCherry, and the photoconvertible FP mEos4b. Our results will allow using FP directionality in studies of molecular and biological processes, but also in development of novel bioengineering and bioelectronics applications.

Institute of Microbiology Czech Academy of Sciences 37333 Nové Hrady Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences 14220 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences 16610 Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences 16610 Prague 6 Czech Republic;

Erratum v

Proc Natl Acad Sci U S A. 2022 Jan 4;119(1):e2121067118. doi: 10.1073/pnas.2121067118. PubMed

Zobrazit více v PubMed

Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C., Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994). PubMed

Greenwald E. C., Mehta S., Zhang J., Genetically encoded fluorescent biosensors illuminate the spatiotemporal regulation of signaling networks. Chem. Rev. 118, 11707–11794 (2018). PubMed PMC

Vrabioiu A. M., Mitchison T. J., Structural insights into yeast septin organization from polarized fluorescence microscopy. Nature 443, 466–469 (2006). PubMed

Bondar A., Lazar J., The G protein G PubMed PMC

Lazar J., Bondar A., Timr S., Firestein S. J., Two-photon polarization microscopy reveals protein structure and function. Nat. Methods 8, 684–690 (2011). PubMed

Han Z., et al. , Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight. PLoS One 9, e113873 (2014). PubMed PMC

Kampmann M., Atkinson C. E., Mattheyses A. L., Simon S. M., Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy. Nat. Struct. Mol. Biol. 18, 643–649 (2011). PubMed PMC

Chan F. T., Kaminski C. F., Kaminski Schierle G. S., HomoFRET fluorescence anisotropy imaging as a tool to study molecular self-assembly in live cells. ChemPhysChem 12, 500–509 (2011). PubMed

Rosell F. I., Boxer S. G., Polarized absorption spectra of green fluorescent protein single crystals: Transition dipole moment directions. Biochemistry 42, 177–183 (2003). PubMed

Shi X., et al. , Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C): Quantitative analysis of FRET in YFP dimers. Biochemistry 46, 14403–14417 (2007). PubMed PMC

Stoner-Ma D., et al. , Proton relay reaction in green fluorescent protein (GFP): Polarization-resolved ultrafast vibrational spectroscopy of isotopically edited GFP. J. Phys. Chem. B 110, 22009–22018 (2006). PubMed

Ansbacher T., et al. , Calculation of transition dipole moment in fluorescent proteins—Towards efficient energy transfer. Phys. Chem. Chem. Phys. 14, 4109–4117 (2012). PubMed

Khrenova M., Topol I., Collins J., Nemukhin A., Estimating orientation factors in the FRET theory of fluorescent proteins: The TagRFP-KFP pair and beyond. Biophys. J. 108, 126–132 (2015). PubMed PMC

Usman A., et al. , Excited-state structure determination of the green fluorescent protein chromophore. J. Am. Chem. Soc. 127, 11214–11215 (2005). PubMed

Inoué S., Shimomura O., Goda M., Shribak M., Tran P. T., Fluorescence polarization of green fluorescence protein. Proc. Natl. Acad. Sci. U.S.A. 99, 4272–4277 (2002). PubMed PMC

Goedhart J., et al. , Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012). PubMed PMC

Myskova J., Rybakova M., Brynda J., Lazar J., mTurquoise2 SG P212121 - Directional optical properties of fluorescent proteins. Protein Data Bank. https://www.rcsb.org/structure/6YLN. Deposited 7 April 2020.

Cormack B. P., Valdivia R. H., Falkow S., FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996). PubMed

Myskova J., Rybakova M., Brynda J., Lazar J., EGFP in neutral pH, Directionality of optical properties of fluorescent proteins. Protein Data Bank. https://www.rcsb.org/structure/unreleased/6YLQ. Deposited 7 April 2020.

Myskova J., Rybakova M., Brynda J., Lazar J., EGFP_in_Acidic_env directionality of optical properties of fluorescent proteins. Protein Data Bank. https://www.rcsb.org/structure/unreleased/6YLP. Deposited 7 April 2020.

Shaner N. C., et al. , Improved monomeric red, orange and yellow fluorescent proteins derived from PubMed

Myskova J., Rybakova M., Brynda J., Lazar J., mCherry. Protein Data Bank. https://www.rcsb.org/structure/6YLM. Deposited 7 April 2020.

Paez-Segala M. G., et al. , Fixation-resistant photoactivatable fluorescent proteins for CLEM. Nat. Methods 12, 215–218 (2015). PubMed PMC

Myskova J., Rybakova M., Brynda J., Lazar J., mEos4b - Directionality of optical properties of fluorescent Proteins. Protein Data Bank. https://www.rcsb.org/structure/unreleased/6YLS. Deposited 7 April 2020.

Ormö M., et al. , Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996). PubMed

Yang F., Moss L. G., Phillips G. N. Jr, The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251 (1996). PubMed

Brejc K., et al. , Structural basis for dual excitation and photoisomerization of the PubMed PMC

Royant A., Noirclerc-Savoye M., Stabilizing role of glutamic acid 222 in the structure of enhanced green fluorescent protein. J. Struct. Biol. 174, 385–390 (2011). PubMed

Arpino J. A., Rizkallah P. J., Jones D. D., Crystal structure of enhanced green fluorescent protein to 1.35 Å resolution reveals alternative conformations for Glu222. PLoS One 7, e47132 (2012). PubMed PMC

De Zitter E., et al. , Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM. Nat. Methods 16, 707–710 (2019). PubMed

Shu X., Shaner N. C., Yarbrough C. A., Tsien R. Y., Remington S. J., Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647 (2006). PubMed

Rocheleau J. V., Edidin M., Piston D. W., Intrasequence GFP in class I MHC molecules, a rigid probe for fluorescence anisotropy measurements of the membrane environment. Biophys. J. 84, 4078–4086 (2003). PubMed PMC

Volkmer A., Subramaniam V., Birch D. J., Jovin T. M., One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys. J. 78, 1589–1598 (2000). PubMed PMC

Oura M., et al. , Polarization-dependent fluorescence correlation spectroscopy for studying structural properties of proteins in living cell. Sci. Rep. 6, 31091 (2016). PubMed PMC

Gotthard G., et al. , Specific radiation damage is a lesser concern at room temperature. IUCrJ 6, 665–680 (2019). PubMed PMC

Gotthard G., von Stetten D., Clavel D., Noirclerc-Savoye M., Royant A., Chromophore isomer stabilization is critical to the efficient fluorescence of cyan fluorescent proteins. Biochemistry 56, 6418–6422 (2017). PubMed

Lelimousin M., et al. , Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield. Biochemistry 48, 10038–10046 (2009). PubMed

Ruiz T., Oldenbourg R., Birefringence of tropomyosin crystals. Biophys. J. 54, 17–24 (1988). PubMed PMC

Owen R. L., Garman E., A new method for predetermining the diffraction quality of protein crystals: Using SOAP as a selection tool. Acta Crystallogr. D Biol. Crystallogr. 61, 130–140 (2005). PubMed

McQuilken M., et al. , Polarized fluorescence microscopy to study cytoskeleton assembly and organization in live cells. Curr. Protocols Cell Biol. 67, 4.29.1–4.29.13 (2015). PubMed PMC

Zhanghao K., et al. , Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy. Nat. Commun. 10, 4694 (2019). PubMed PMC

Gather M. C., Yun S. H., Single-cell biological lasers. Nat. Photonics 5, 406–410 (2011).

Cinelli R. A., et al. , Green fluorescent proteins as optically controllable elements in bioelectronics. Appl. Phys. Lett. 79, 3353–3355 (2001).

Gerlach M., Mueller U., Weiss M. S., The MX beamlines BL14. 1-3 at BESSY II. J. Large-Scale Res. 2, 47 (2016).

Kabsch W., Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010). PubMed PMC

Winn M. D., et al. , Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011). PubMed PMC

Vagin A., Teplyakov A., Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010). PubMed

Vagin A. A., et al. , REFMAC5 dictionary: Organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60, 2184–2195 (2004). PubMed

Schrodinger , The PyMOL Molecular Graphics System (Schrodinger LLC, 2010), Version 1.0.

Pettersen E., et al. , UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 223, 1605–1612 (2004). PubMed

Thévenaz P., Ruttimann U. E., Unser M., A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27–41 (1998). PubMed

A genetically encoded nanobody sensor reveals conformational diversity in β-arrestins orchestrated by distinct seven transmembrane receptors

Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools