Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.

Department of Cell and Molecular Biology Uppsala University Uppsala Sweden

Escola Superior de Tecnologia do Barreiro Instituto Politécnico de Setúbal Portugal

Faculty of Science Charles University Prague Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences Czech Republic

Institute of Molecular Science Universitat de València Paterna Spain

Zobrazit více v PubMed

Adhikary R, Zimmermann J, Dawson PE, Romesberg FE. Temperature dependence of CN and SCN IR absorptions facilitates their interpretation and use as probes of proteins. Anal Chem. 2015;87:11561–7. PubMed

Adhikary R, Zimmermann J, Romesberg FE. Transparent window vibrational probes for the characterization of proteins with high structural and temporal resolution. Chem Rev. 2017;117:1927–69. PubMed

Andrikopoulos PC, Liu Y, Picchiotti A, Lenngren N, Kloz M, Chaudhari AS, et al. Femtosecond‐to‐nanosecond dynamics of flavin mononucleotide monitored by stimulated Raman spectroscopy and simulations. Phys Chem Chem Phys. 2020;22:6538–52. PubMed

Andrikopoulos PC, Chaudhari AS, Liu Y, Konold PE, Kennis JTM, Schneider B, et al. QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors. Phys Chem Chem Phys. 2021;23:13934–50. PubMed PMC

Arrondo JLR, Muga A, Castresana J, Goñi FM. Quantitative studies of the structure of proteins in solution by fourier‐transform infrared spectroscopy. Prog Biophys Mol Biol. 1993;59:23–56. PubMed

Bannister S, Böhm E, Zinn T, Hellweg T, Kottke T. Arguments for an additional long‐lived intermediate in the photocycle of the full‐length aureochrome 1c receptor: a time‐resolved small‐angle X‐ray scattering study. Struct Dyn. 2019;6:034701. PubMed PMC

Barth A. Infrared spectroscopy of proteins. Biochim Biophys Acta Bioenergetics. 2007;1767:1073–101. PubMed

Berntsson O, Rodriguez R, Henry L, Panman MR, Hughes AJ, Einholz C, et al. Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions. Sci Adv. 2019;5:eaaw1531. PubMed PMC

Blankenburg L, Schroeder L, Habenstein F, Blasiak B, Kottke T, Bredenbeck J. Following local light‐induced structure changes and dynamics of the photoreceptor PYP with the thiocyanate IR label. Phys Chem Chem Phys. 2019;21:6622–34. PubMed

Bocola M, Schwaneberg U, Jaeger K‐E, Krauss U. Light‐induced structural changes in a short light, oxygen, voltage (LOV) protein revealed by molecular dynamics simulations—implications for the understanding of LOV photoactivation. Front Mol Biosci. 2015;2. PubMed PMC

Corchnoy SB, Swartz TE, Lewis JW, Szundi I, Briggs WR, Bogomolni RA. Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of Phototropin 1. J Biol Chem. 2003;278:724–31. PubMed

Cousin SF, Kadeřávek P, Bolik‐Coulon N, Gu Y, Charlier C, Carlier L, et al. Time‐resolved protein side‐chain motions unraveled by high‐resolution relaxometry and molecular dynamics simulations. J Am Chem Soc. 2018;140:13456–65. PubMed

Davis CM, Dyer RB. WW domain folding complexity revealed by infrared spectroscopy. Biochemistry. 2014;53:5476–84. PubMed PMC

Davis CM, Cooper AK, Dyer RB. Fast helix formation in the B domain of protein a revealed by site‐specific infrared probes. Biochemistry. 2015;54:1758–66. PubMed PMC

Davis CM, Zanetti‐Polzi L, Gruebele M, Amadei A, Dyer RB, Daidone I. A quantitative connection of experimental and simulated folding landscapes by vibrational spectroscopy. Chem Sci. 2018;9:9002–11. PubMed PMC

Fafarman AT, Webb LJ, Chuang JI, Boxer SG. Site‐specific conversion of cysteine thiols into thiocyanate creates an IR probe for electric fields in proteins. J Am Chem Soc. 2006;128:13356–7. PubMed PMC

Fayer MD. Fast protein dynamics probed with infrared vibrational Echo experiments. Annu Rev Phys Chem. 2001;52:315–56. PubMed

First JT, Slocum JD, Webb LJ. Quantifying the effects of hydrogen bonding on nitrile frequencies in GFP: beyond solvent exposure. J Phys Chem B. 2018;122:6733–43. PubMed

Getahun Z, Huang CY, Wang T, De Leon B, DeGrado WF, Gai F. Using nitrile‐derivatized amino acids as infrared probes of local environment. J Am Chem Soc. 2003;125:405–11. PubMed

Gil AA, Laptenok SP, French JB, Iuliano JN, Lukacs A, Hall CR, et al. Femtosecond to millisecond dynamics of light induced Allostery in the Avena sativa LOV domain. J Phys Chem B. 2017;121:1010–9. PubMed PMC

Goett‐Zink L, Klocke JL, Bögeholz LAK, Kottke T. In‐cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells. J Biol Chem. 2020;295:11729–41. PubMed PMC

Hagen SJ, Eaton WA. Two‐state expansion and collapse of a polypeptide 1 1Edited by A. R Fersht. J Mol Biol. 2000;301:1019–27. PubMed

Hall CR, Tolentino Collado J, Iuliano JN, Gil AA, Adamczyk K, Lukacs A, et al. Site‐specific protein dynamics probed by ultrafast infrared spectroscopy of a noncanonical amino acid. J Phys Chem B. 2019;123:9592–7. PubMed

Harper SM, Neil LC, Day IJ, Hore PJ, Gardner KH. Conformational changes in a photosensory LOV domain monitored by time‐resolved NMR spectroscopy. J Am Chem Soc. 2004;126:3390–1. PubMed

Hasegawa K, Masuda S, Ono T‐A. Spectroscopic analysis of the dark relaxation process of a photocycle in a sensor of blue light using FAD (BLUF) protein Slr1694 of the Cyanobacterium synechocystis sp. PCC6803. Plant Cell Physiol. 2005;46:136–46. PubMed

Heintz U, Schlichting I. Blue light‐induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence. Elife. 2016;5:e11860. PubMed PMC

Henry ER, Best RB, Eaton WA. Comparing a simple theoretical model for protein folding with all‐atom molecular dynamics simulations. Proc Natl Acad Sci U S A. 2013;110:17880–5. PubMed PMC

Herman E, Kottke T. Allosterically regulated unfolding of the A’α helix exposes the dimerization site of the blue‐light‐sensing Aureochrome‐LOV domain. Biochemistry. 2015;54:1484–92. PubMed

Herman E, Sachse M, Kroth PG, Kottke T. Blue‐light‐induced unfolding of the Jα helix allows for the dimerization of Aureochrome‐LOV from the diatom Phaeodactylum tricornutum. Biochemistry. 2013;52:3094–101. PubMed

Ihalainen JA, Paoli B, Muff S, Backus EHG, Bredenbeck J, Woolley GA, et al. Helix folding in the presence of structural constraints. Proc Natl Acad Sci U S A. 2008;105:9588–93. PubMed PMC

Iuliano JN, Gil AA, Laptenok SP, Hall CR, Tolentino Collado J, Lukacs A, et al. Variation in LOV photoreceptor activation dynamics probed by time‐resolved infrared spectroscopy. Biochemistry. 2018;57:620–30. PubMed PMC

Jas GS, Kuczera K. Helix–coil transition courses through multiple pathways and intermediates: fast kinetic measurements and dimensionality reduction. J Phys Chem B. 2018;122:10806–16. PubMed

Kennis JTM, van Stokkum IHM, Crosson S, Gauden M, Moffat K, van Grondelle R. The LOV2 domain of Phototropin: a reversible photochromic switch. J Am Chem Soc. 2004;126:4512–3. PubMed

Konold PE, Mathes T, Weiβenborn J, Groot ML, Hegemann P, Kennis JTM. Unfolding of the C‐terminal Jα helix in the LOV2 photoreceptor domain observed by time‐resolved vibrational spectroscopy. J Phys Chem Lett. 2016;7:3472–6. PubMed

Kottke T, Lorenz‐Fonfria VA, Heberle J. The grateful infrared: sequential protein structural changes resolved by infrared difference spectroscopy. J Phys Chem B. 2017;121:335–50. PubMed

Kottke T, Xie A, Larsen DS, Hoff WD. Photoreceptors take charge: emerging principles for light sensing. Annu Rev Biophys. 2018;47:291–313. PubMed

Kraskov A, von Sass J, Nguyen AD, Hoang TO, Buhrke D, Katz S, et al. Local electric field changes during the photoconversion of the bathy phytochrome Agp2. Biochemistry. 2021;60:2967–77. PubMed

Krause BS, Kaufmann JCD, Kuhne J, Vierock J, Huber T, Sakmar TP, et al. Tracking pore hydration in Channelrhodopsin by site‐directed infrared‐active azido probes. Biochemistry. 2019;58:1275–86. PubMed

Krimm S, Bandekar J. Vibrational Spectroscopy and Conformation of Peptides, Polypeptides, and Proteins. Advances in Protein Chemistry. Advances in Protein Chemistry. Elsevier Academic Press. Volume 38; 1986. p. 181–364. PubMed

Kulkarni P, Solomon TL, He Y, Chen Y, Bryan PN, Orban J. Structural metamorphism and polymorphism in proteins on the brink of thermodynamic stability. Protein Sci. 2018;27:1557–67. PubMed PMC

Kurttila M, Stucki‐Buchli B, Rumfeldt J, Schroeder L, Häkkänen H, Liukkonen A, et al. Site‐by‐site tracking of signal transduction in an azidophenylalanine‐labeled bacteriophytochrome with step‐scan FTIR spectroscopy. Phys Chem Chem Phys. 2021;23:5615–28. PubMed

Lebedev N, Zayner JP, Sosnick TR. Factors that control the chemistry of the LOV domain photocycle. PLoS ONE. 2014;9. PubMed PMC

Lee SJ, Kim TW, Kim JG, Yang C, Yun SR, Kim C, et al. Light‐induced protein structural dynamics in bacteriophytochrome revealed by time‐resolved x‐ray solution scattering. Sci Adv. 2022;8:eabm6278. PubMed PMC

Lim M, Hamm P, Hochstrasser RM. Protein fluctuations are sensed by stimulated infrared echoes of the vibrations of carbon monoxide and azide probes. Proc Natl Acad Sci U S A. 1998;95:15315–20. PubMed PMC

Liu Y, Chaudhari AS, Chatterjee A, Andrikopoulos PC, Picchiotti A, Rebarz M, et al. Sub‐millisecond photoinduced dynamics of free and EL222‐bound FMN by stimulated Raman and visible absorption spectroscopies. Biomolecules. 2023;13. PubMed PMC

Löffler JG, Deniz E, Feid C, Franz VG, Bredenbeck J. Versatile vibrational energy sensors for proteins. Angew Chem Int Ed. 2022;61. PubMed PMC

Lorenz‐Fonfria VA. Infrared difference spectroscopy of proteins: from bands to bonds. Chem Rev. 2020;120:3466–576. PubMed

Lórenz‐Fonfría VA, Kandori H. Bayesian maximum entropy (two‐dimensional) lifetime distribution reconstruction from time‐resolved spectroscopic data. Appl Spectrosc. 2016;61:428–43. PubMed

Ma H, Gruebele M. Kinetics are probe‐dependent during downhill folding of an engineered 6‐85 protein. Proc Natl Acad Sci U S A. 2005;102:2283–7. PubMed PMC

Maia RNA, Ehrenberg D, Oldemeyer S, Knieps‐Grünhagen E, Krauss U, Heberle J. Real‐time tracking of proton transfer from the reactive cysteine to the flavin chromophore of a Photosensing light oxygen voltage protein. J Am Chem Soc. 2021;143:12535–42. PubMed

Manandhar M, Chun E, Romesberg FE. Genetic code expansion: inception, development, commercialization. J Am Chem Soc. 2021;143:4859–78. PubMed

Milles S, Tyagi S, Banterle N, Koehler C, VanDelinder V, Plass T, et al. Click strategies for single‐molecule protein fluorescence. J Am Chem Soc. 2012;134:5187–95. PubMed

Miyake‐Stoner SJ, Miller AM, Hammill JT, Peeler JC, Hess KR, Mehl RA, et al. Probing protein folding using site‐specifically encoded unnatural amino acids as FRET donors with tryptophan. Biochemistry. 2009;48:5953–62. PubMed

Miyamori T, Nakasone Y, Hitomi K, Christie JM, Getzoff ED, Terazima M. Reaction dynamics of the UV‐B photosensor UVR8. Photochem Photobiol Sci. 2015;14:995–1004. PubMed

Nagarajan S, Taskent‐Sezgin H, Parul D, Carrico I, Raleigh DP, Dyer RB. Differential ordering of the protein backbone and side chains during protein folding revealed by site‐specific recombinant infrared probes. J Am Chem Soc. 2011;133:20335–40. PubMed PMC

Nakasone Y, Eitoku T, Matsuoka D, Tokutomi S, Terazima M. Dynamics of conformational changes of Arabidopsis Phototropin 1 LOV2 with the linker domain. J Mol Biol. 2007;367:432–42. PubMed

Nash AI, McNulty R, Shillito ME, Swartz TE, Bogomolni RA, Luecke H, et al. Structural basis of photosensitivity in a bacterial light‐oxygen‐voltage/helix‐turn‐helix (LOV‐HTH) DNA‐binding protein. Proc Natl Acad Sci U S A. 2011;108:9449–54. PubMed PMC

Oh K‐I, Lee J‐H, Joo C, Han H, Cho M. β‐Azidoalanine as an IR probe: application to amyloid Aβ(16‐22) aggregation. J Phys Chem B. 2008;112:10352–7. PubMed

Porter LL, Looger LL. Extant fold‐switching proteins are widespread. Proc Natl Acad Sci U S A. 2018;115:5968–73. PubMed PMC

Prigozhin MB, Chao SH, Sukenik S, Pogorelov TV, Gruebele M. Mapping fast protein folding with multiple‐site fluorescent probes. Proc Natl Acad Sci U S A. 2015;112:7966–71. PubMed PMC

Schultz KC, Supekova L, Ryu Y, Xie J, Perera R, Schultz PG. A genetically encoded infrared probe. J Am Chem Soc. 2006;128:13984–5. PubMed

Spexard M, Immeln D, Thöing C, Kottke T. Infrared spectrum and absorption coefficient of the cofactor flavin in water. Vibrat Spectrosc. 2011;57:282–7.

Stock G, Hamm P. A non‐equilibrium approach to allosteric communication. Philos Trans R Soc B Biol Sci. 2018;373:20170187. PubMed PMC

van Stokkum IHM, Larsen DS, van Grondelle R. Global and target analysis of time‐resolved spectra. Biochim Biophys Acta Bioenergetics. 2004;1657:82–104. PubMed

Sukenik S, Pogorelov TV, Gruebele M. Can local probes go global? A joint experiment‐simulation analysis of lambda(6‐85) folding. J Phys Chem Lett. 2016;7:1960–5. PubMed

Suydam IT, Boxer SG. Vibrational stark effects calibrate the sensitivity of vibrational probes for electric fields in proteins. Biochemistry. 2003;42:12050–5. PubMed

Takakado A, Nakasone Y, Terazima M. Photoinduced dimerization of a photosensory DNA‐binding protein EL222 and its LOV domain. Phys Chem Chem Phys. 2017;19:24855–65. PubMed

Takakado A, Nakasone Y, Terazima M. Sequential DNA binding and dimerization processes of the photosensory protein EL222. Biochemistry. 2018;57:1603–10. PubMed

Takala H, Lehtivuori HK, Berntsson O, Hughes A, Nanekar R, Niebling S, et al. On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome. J Biol Chem. 2018;293:8161–72. PubMed PMC

Taskent‐Sezgin H, Chung J, Banerjee PS, Nagarajan S, Dyer RB, Carrico I, et al. Azidohomoalanine: a conformationally sensitive IR probe of protein folding, protein structure, and electrostatics. Angew Chem Int Ed Engl. 2010;49:7473–5. PubMed PMC

Thielges MC. Transparent window 2D IR spectroscopy of proteins. J Chem Phys. 2021;155:40903. PubMed PMC

Tolentino Collado J, Iuliano JN, Pirisi K, Jewlikar S, Adamczyk K, Greetham GM, et al. Unraveling the photoactivation mechanism of a light‐activated adenylyl cyclase using ultrafast spectroscopy coupled with unnatural amino acid mutagenesis. ACS Chem Biol. 2022;17:2643–54. PubMed PMC

Uzawa T, Akiyama S, Kimura T, Takahashi S, Ishimori K, Morishima I, et al. Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of ‐helical content and compactness. Proc Natl Acad Sci U S A. 2004;101:1171–6. PubMed PMC

Vaidya AT, Chen CH, Dunlap JC, Loros JJ, Crane BR. Structure of a light‐activated LOV protein dimer that regulates transcription. Sci Signal. 2011;4:ra50‐ra50. PubMed PMC

Weaver JB, Kozuch J, Kirsh JM, Boxer SG. Nitrile infrared intensities characterize electric fields and hydrogen bonding in protic, aprotic, and protein environments. J Am Chem Soc. 2022;144:7562–7. PubMed PMC

Yang W‐J, Griffiths PR, Byler DM, Susi H. Protein conformation by infrared spectroscopy: resolution enhancement by Fourier self‐deconvolution. Appl Spectrosc. 2016;39:282–7.

Ye S, Huber T, Vogel R, Sakmar TP. FTIR analysis of GPCR activation using azido probes. Nat Chem Biol. 2009;5:397–9. PubMed PMC

Ye S, Zaitseva E, Caltabiano G, Schertler GF, Sakmar TP, Deupi X, et al. Tracking G‐protein‐coupled receptor activation using genetically encoded infrared probes. Nature. 2010;464:1386–9. PubMed

Zhang J, Wang L, Zhang J, Zhu J, Pan X, Cui Z, et al. Identifying and modulating accidental Fermi resonance: 2D IR and DFT study of 4‐azido‐l‐phenylalanine. J Phys Chem B. 2018;122:8122–33. PubMed

Zoltowski BD, Nash AI, Gardner KH. Variations in protein‐flavin hydrogen bonding in a light, oxygen, voltage domain produce non‐Arrhenius kinetics of adduct decay. Biochemistry. 2011;50:8771–9. PubMed PMC

Zoltowski BD, Motta‐Mena LB, Gardner KH. Blue light‐induced dimerization of a bacterial LOV‐HTH DNA‐binding protein. Biochemistry. 2013;52:6653–61. PubMed PMC

Light-dependent flavin redox and adduct states control the conformation and DNA-binding activity of the transcription factor EL222

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies