Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Department of Physics and Astronomy Vrije Universiteit Amsterdam Amsterdam 1081 HV The Netherlands

Departments of Anatomy and Structural Biology Albert Einstein College of Medicine Bronx NY 10461 USA

ELI Beamlines Institute of Physics Na Slovance 2 182 21 Praha 8 Czech Republic

Institut für Chemie Technische Universität Berlin Sekr PC 14 Straße des 17 Juni 135 Berlin D 10623 Germany

Medicum Faculty of Medicine University of Helsinki Helsinki 00290 Finland

Present address School of Applied and Engineering Physics Cornell University Ithaca NY 14853 USA

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Tsien RY. Constructing and Exploiting the Fluorescent Protein Paintbox (Nobel Lecture) Angew. Chem. Int. Ed. 2009;48:5612–5626. doi: 10.1002/anie.200901916. PubMed DOI

Weissleder R, Pittet MJ. Imaging in the era of molecular oncology. Nature. 2008;452:580–589. doi: 10.1038/nature06917. PubMed DOI PMC

Shu X, et al. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science. 2009;324:804–807. doi: 10.1126/science.1168683. PubMed DOI PMC

Filonov GS, et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat. Biotechnol. 2011;29:757–761. doi: 10.1038/nbt.1918. PubMed DOI PMC

Shcherbakova DM, Verkhusha VV. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods. 2013;10:751–754. doi: 10.1038/nmeth.2521. PubMed DOI PMC

Shcherbakova DM, Baloban M, Verkhusha VV. Near-infrared fluorescent proteins engineered from bacterial phytochromes. Curr. Opin. Chem. Biol. 2015;27:52–63. doi: 10.1016/j.cbpa.2015.06.005. PubMed DOI PMC

Shcherbakova, D. M., Shemetov, A. A., Kaberniuk, A. A. & Verkhusha, V. V. Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools. Annu. Rev. Biochem.84, 519–550 (2015). PubMed PMC

Shcherbakova DM, et al. Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nat. Commun. 2016;7:1–12. doi: 10.1038/ncomms12405. PubMed DOI PMC

Matlashov, M. E. et al. A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nat. Commun. 11, 239 (2020). PubMed PMC

Auldridge ME, Satyshur KA, Anstrom DM, Forest KT. Structure-guided engineering enhances a phytochrome-based infrared fluorescent protein. J. Biol. Chem. 2012;287:7000–7009. doi: 10.1074/jbc.M111.295121. PubMed DOI PMC

Baird GS, Zacharias DA, Tsien RY. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl Acad. Sci. USA. 1999;96:11241–11246. doi: 10.1073/pnas.96.20.11241. PubMed DOI PMC

Barondeau DP, Putnam CD, Kassmann CJ, Tainer JA, Getzoff ED. Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures. Proc. Natl Acad. Sci. USA. 2003;100:12111–12116. doi: 10.1073/pnas.2133463100. PubMed DOI PMC

Zhang L, Patel HN, Lappe JW, Wachter RM. Reaction progress of chromophore biogenesis in green fluorescent protein. J. Am. Chem. Soc. 2006;128:4766–4772. doi: 10.1021/ja0580439. PubMed DOI

Craggs TD. Green fluorescent protein: structure, folding and chromophore maturation. Chem. Soc. Rev. 2009;38:2865–2875. doi: 10.1039/b903641p. PubMed DOI

Shemetov AA, Oliinyk OS, Verkhusha VV. How to increase brightness of near-infrared fluorescent proteins in mammalian cells. Cell Chem. Biol. 2017;24:758–766.e3. doi: 10.1016/j.chembiol.2017.05.018. PubMed DOI PMC

Kloz M, Weißenborn J, Polívka T, Frank HA, Kennis JTM. Spectral watermarking in femtosecond stimulated Raman spectroscopy: Resolving the nature of the carotenoid S∗ state. Phys. Chem. Chem. Phys. 2016;18:14619–14628. doi: 10.1039/C6CP01464J. PubMed DOI

Hontani Y, et al. The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy. Phys. Chem. Chem. Phys. 2016;18:24729–24736. doi: 10.1039/C6CP05240A. PubMed DOI

Hontani Y, et al. Molecular origin of photoprotection in cyanobacteria probed by watermarked femtosecond stimulated Raman spectroscopy. J. Phys. Chem. Lett. 2018;9:1788–1792. doi: 10.1021/acs.jpclett.8b00663. PubMed DOI PMC

Hontani Y, et al. Strong pH-dependent near-infrared fluorescence in a microbial rhodopsin reconstituted with a red-shifting retinal analogue. J. Phys. Chem. Lett. 2018;9:6469–6474. doi: 10.1021/acs.jpclett.8b02780. PubMed DOI PMC

Pižl M, et al. Time-resolved femtosecond stimulated Raman spectra and DFT anharmonic vibrational analysis of an electronically excited rhenium photosensitizer. J. Phys. Chem. A. 2020;124:1253–1265. doi: 10.1021/acs.jpca.9b10840. PubMed DOI

Hontani Y, et al. Dual photoisomerization on distinct potential energy surfaces in a UV absorbing rhodopsin. J. Am. Chem. Soc. 2020;142:11464–11473. doi: 10.1021/jacs.0c03229. PubMed DOI PMC

Artes Vivancos, J. M. et al. Unraveling the excited-state dynamics and light-harvesting functions of xanthophylls in light-harvesting complex II using femtosecond stimulated Raman spectroscopy. J. Am. Chem. Soc. 10.1021/jacs.0c04619 (2020). PubMed PMC

Shcherbakova DM, et al. Molecular basis of spectral diversity in near-infrared phytochrome-based fluorescent proteins. Chem. Biol. 2015;22:1540–1551. doi: 10.1016/j.chembiol.2015.10.007. PubMed DOI PMC

Shcherbakova DM, Cammer Cox, Huisman N, Verkhusha TM, Hodgson VV. L. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat. Chem. Biol. 2018;14:591–600. doi: 10.1038/s41589-018-0044-1. PubMed DOI PMC

Monakhov MV, et al. Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins. ACS Chemical Neuroscience. 2020;11:3523–3531. doi: 10.1021/acschemneuro.0c00046. PubMed DOI

Wagner JR, Brunzelle JS, Forest KT, Vierstra RD. A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome. Nature. 2005;438:325–331. doi: 10.1038/nature04118. PubMed DOI

Wagner JR, Zhang J, Brunzelle JS, Vierstra RD, Forest KT. High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution. J. Biol. Chem. 2007;282:12298–12309. doi: 10.1074/jbc.M611824200. PubMed DOI

Yang X, Stojkovic EA, Kuk J, Moffat K. Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion. Proc. Natl Acad. Sci. USA. 2007;104:12571–12576. doi: 10.1073/pnas.0701737104. PubMed DOI PMC

Lamparter T, Michael N, Mittmann F, Esteban B. Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site. Proc. Natl Acad. Sci. USA. 2002;99:11628–11633. doi: 10.1073/pnas.152263999. PubMed DOI PMC

Lamparter T, et al. The Biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of agrobacterium phytochrome Agp1. Biochemistry. 2004;43:3659–3669. doi: 10.1021/bi035693l. PubMed DOI

Baloban M, et al. Designing brighter near-infrared fluorescent proteins: Insights from structural and biochemical studies. Chem. Sci. 2017;8:4546–4557. doi: 10.1039/C7SC00855D. PubMed DOI PMC

Buhrke D, et al. Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins. Sci. Rep. 2019;9:1866. doi: 10.1038/s41598-018-38433-2. PubMed DOI PMC

Margulies L, Toporowicz M. Resonance Raman study of model compounds of the phytochrome chromophore. 2. Biliverdin dimethyl ester. J. Am. Chem. Soc. 1984;106:7331–7336. doi: 10.1021/ja00336a007. DOI

Hsieh YZ, Morris MD. Resonance Raman spectroscopic study of bilirubin hydrogen bonding in solutions and in the albumin complex. J. Am. Chem. Soc. 1988;110:62–67. doi: 10.1021/ja00209a009. DOI

Holt RE, Farrens DL, Song PS, Cotton TM. Surface-enhanced resonance Raman scattering (SERRS) spectroscopy applied to phytochrome and its model compounds. I. Biliverdin photoisomers. J. Am. Chem. Soc. 1989;111:9156–9162. doi: 10.1021/ja00208a003. DOI

Iturraspe JB, Bari S, Frydman B. Total synthesis of ‘extended’ biliverdins. The relation between their conformation and their spectroscopic properties. J. Am. Chem. Soc. 1989;111:1525–1527. doi: 10.1021/ja00186a072. DOI

Wagner JR, et al. Mutational analysis of Deinococcus radiodurans bacteriophytochrome reveals key amino acids necessary for the photochromicity and proton exchange cycle of phytochromes. J. Biol. Chem. 2008;283:12212–12226. doi: 10.1074/jbc.M709355200. PubMed DOI PMC

Matute RA, Contreras R, González L. Time-dependent DFT on phytochrome chromophores: a way to the right conformer. J. Phys. Chem. Lett. 2010;1:796–801. doi: 10.1021/jz900432m. DOI

Zienicke, B. et al. Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr *. 10.1074/jbc.M113.479535 (2013). PubMed PMC

Velazquez Escobar F, et al. Protonation-dependent structural heterogeneity in the chromophore binding site of cyanobacterial phytochrome Cph1. J. Phys. Chem. B. 2017;121:47–57. doi: 10.1021/acs.jpcb.6b09600. PubMed DOI

Velazquez Escobar F, et al. Structural parameters controlling the fluorescence properties of phytochromes. Biochemistry. 2014;53:20–29. doi: 10.1021/bi401287u. PubMed DOI

Kneip C, et al. Protonation state and structural changes of the tetrapyrrole chromophore during the P(r) → P(fr) phototransformation of phytochrome: a resonance raman spectroscopic study. Biochemistry. 1999;38:15185–15192. doi: 10.1021/bi990688w. PubMed DOI

Mroginski MA, et al. Chromophore structure of cyanobacterial phytochrome Cph1 in the Pr state: reconciling structural and spectroscopic data by QM/MM calculations. Biophys. J. 2009;96:4153–4163. doi: 10.1016/j.bpj.2009.02.029. PubMed DOI PMC

von Stetten D, et al. Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation. J. Biol. Chem. 2007;282:2116–2123. doi: 10.1074/jbc.M608878200. PubMed DOI

Hontani Y, et al. Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics. Sci. Rep. 2016;6:1–12. doi: 10.1038/srep37362. PubMed DOI PMC

Snellenburg JJ, Laptenok SP, Seger R, Mullen KM, van Stokkum IHM. Glotaran: a Java-based graphical user interface for the R package TIMP. J. Stat. Softw. 2012;49:1–2. doi: 10.18637/jss.v049.i03. DOI

Van Stokkum IHM, Larsen DS, Van Grondelle R. Global and target analysis of time-resolved spectra. Biochim. Biophys. Acta - Bioenerg. 2004;1657:82–104. doi: 10.1016/j.bbabio.2004.04.011. PubMed DOI

Kennis JT, Groot ML. Ultrafast spectroscopy of biological photoreceptors. Curr. Opin. Struct. Biol. 2007;17:623–630. doi: 10.1016/j.sbi.2007.09.006. PubMed DOI

Toh KC, Stojković EA, Van Stokkum IHM, Moffat K, Kennis JTM. Fluorescence quantum yield and photochemistry of bacteriophytochrome constructs. Phys. Chem. Chem. Phys. 2011;13:11985–11997. doi: 10.1039/c1cp00050k. PubMed DOI

Brooks BR, et al. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 1983;4:187–217. doi: 10.1002/jcc.540040211. DOI

Foloppe N, Alexander D, MacKerell J. All‐atom empirical force field for nucleic acids: I. Parameter optimization based on small molecule and condensed phase macromolecular target data. J. Comput. Chem. 2000;21:86–104. doi: 10.1002/(SICI)1096-987X(20000130)21:2<86::AID-JCC2>3.0.CO;2-G. DOI

Senn HM, Thiel W. QM/MM methods for biomolecular systems. Angew. Chem. Int. Ed. 2009;48:1198–1229. doi: 10.1002/anie.200802019. PubMed DOI

Billeter SR, Turner AJ, Thiel W. Linear scaling geometry optimisation and transition state search in hybrid delocalised internal coordinates. Phys. Chem. Chem. Phys. 2000;2:2177–2186. doi: 10.1039/a909486e. DOI

Sherwood P, et al. QUASI: a general purpose implementation of the QM/MM approach and its application to problems in catalysis. J. Mol. Struct. THEOCHEM. 2003;632:1–28. doi: 10.1016/S0166-1280(03)00285-9. DOI

Mroginski MA, Mark F, Thiel W, Hildebrandt P. Quantum mechanics/molecular mechanics calculation of the Raman spectra of the phycocyanobilin chromophore in α-C-phycocyanin. Biophys. J. 2007;93:1885–1894. doi: 10.1529/biophysj.107.108878. PubMed DOI PMC

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