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Two-Photon Excitation of Flavins and Flavoproteins with Classical and Quantum Light Juan P. Villabona-Monsalve, Oleg Varnavski, Bruce A. Palfey, and Theodore Goodson III J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08515 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 22, 2018

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Journal of the American Chemical Society

Two-Photon Excitation of Flavins and Flavoproteins with Classical and Quantum Light. Juan P. Villabona-Monsalve,† Oleg Varnavski,† Bruce A. Palfey,‡ and Theodore Goodson III†,* †

Department of Chemistry, University of Michigan, Ann Arbor, MI 48109. Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, United States ‡

Supporting Information Placeholder ABSTRACT: In this contribution, the entangled two-photon absorption (ETPA) process on naturally occurring flavoproteins was studied. Low temperature responsive protein (LOT6P) and b-Type dihydroorotate dehydrogenase (DHOD B) which possess flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) chromophores embedded in the protein environment were investigated. ETPA cross-section was measured and we found that increases when going from aqueous solution of the free flavin chromophore to the chromophore embedded in the protein. This enhancement is particularly evident when entangled photons are used as excitation light compared to classical light. Our results prove the potential of ETPA as a sensing technique for fluorescent proteins even for those whose classical TPA cross-section is small compared to well-known fluorescent proteins.

Two-photon absorption (TPA) has been used to excite fluorescent proteins since shortly after the discovery of the green fluorescent protein.1-2 Experimental techniques like two-photon laser scanning microscopy (TPLSM)2 and TPA-Förster resonance energy transfer (TPA-FRET)3, have utilized the fluorescence induced in proteins when excited by TPA. Some of the advantages of TPA excitation are photobleaching reduction, deep penetration and three-dimensional high resolution. Classical TPA activity of a molecule is quantified by the TPA cross-section, δTPA. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), shown in Figure 1, are usual cofactors of proteins having enzymatic,4-6 photosensing7-8 and photorepairing activity.9 Two common flavoproteins are DNA photolyase10 and cryptochromes.11 Most of the research on the photophysics of flavoproteins have focused on the steady-state and time resolved (fs to ns) UV-Vis spectroscopy.12-18 Currently, the fluorescence lifetime, the excited state lifetime and the photoinduced electron transfer rate constants are well-known properties of FMN and FAD, free and protein-encaged, in a variety of oxidation states and at different pH values in solution.9, 13-14, 16-24 Nevertheless, two-photon absorption on flavins and flavoproteins has not been studied extensively.2, 25-28 On the search of new light sources to induced TPA, entangled photons were proposed theoretically29-31 and later probe experimentally32-36 to be efficient in two-photon excitation of different organic chromophores. Entangled two-photon absorption (ETPA) has emerged as a new technique to study non-linear properties of organic molecules. In addition to a dramatic decrease of the input photon flux necessary to induce TPA, ETPA also presents the advantage of a linear dependence on the signal, contrary to the quadratic dependence on classical (random) TPA. Because of the ultralow intensity of entangled photons, this light source fills the requirements to be a powerful tool for the two-photon excitation of biological samples ACS Paragon Plus Environment

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where a decrease of photobleaching and photodamage of the sample is required. In this work we studied the ETPA process on FMN, FAD and two flavoproteins, LOT6P and DHOD B (see Figure 1). We determined the ETPA cross-section of the flavin chromophores free and protein-bound, both in aqueous solution, pH = 7.4. This is the first time ETPA is studied for the biologically relevant molecules FMN and FAD and for one group of biomolecules, represented by the two evaluated flavoproteins LOT6P and DHOD B. LOT6P folds in a similar way to flavodoxin, has a low temperature response and is classified as a quinone oxidoreductase.5 It is a homodimer with FMN cofactors on each subunit. DHOD B (1B-Type dihydroorotate dehydrogenase) is a dimer of heterodimers, each heterodimer has the protein subunits PyrDB, which has FMN and PyrK, which has FAD.6, 37 FMN and FAD are stabilized on the protein pocket by hydrogen bonding with well-identified protein amino acids.4, 37

Figure 1. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and related proteins studied here. a) Molecular structures for FMN and FAD. b) FMN bound to DHOD B. c) FMN bound to LOT6P. d) FAD bound to DHOD B. Blue dashed lines represent H-bonds stabilizing FMN or FAD. LOT6P FMN and DHOD B FMN and FAD images were taken from 1T0I.pdb4 and 1EP1.pdb37 files on the protein data bank.38-39 The molecular property that quantifies the ETPA at a given wavelength is the ETPA cross-section, σETPA, whose units are cm2 molecule-1. Due to quantum entanglement, induced by spontaneous parametric downconversion (SPDC), the ETPA rate depends linearly on the incident photon flux rather than quadratic as occurs for classical TPA.31, 33 Thus, a plot of the absorbed photon rate as a function of the input photon flux, fits to a linear equation and the slope of that fitting is directly proportional to σETPA.34 The linear dependence of the absorbed photon rate on the input photon flux, and the fact that the energy of the entangled photon pair allows to access electronic states, point out the occurrence of ETPA as have been probed for a variety of molecular systems.32-33, 36, 40 Figure 2 shows the absorption and fluorescence (one and two-photon excitation) spectra obtained for FAD, DHOD B and LOT6P. For FAD the absorption spectrum presents the S0 → S1 centered at 450 nm with a weak shoulder at ~ 472 nm and the S0 → S2 band is centered at 375 nm. For DHOD B, having the FMN and FAD cofactors, the S0 → S1 transition is T. Goodson III

2 ACS Paragon Plus Environment

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centered at 454 nm accompanied by two weak shoulders at 424 and 474 nm and the S0 → S2 transition is centered at 378 nm. Finally, LOT6P, having only the FMN cofactor, shows the S0 → S1 transition centered at 450 nm along with a ~ 480 nm weak shoulder and the S0 → S2 transition centered at 360 nm. For these flavoproteins the shoulder at ~480 nm has been identified as characteristic of flavin chromophores binding to proteins.5 The appearance of vibrational structure in the absorption spectrum is related with the reduction of the conformational variations of flavins when embedded in the protein.28, 41

Figure 2. Normalized absorption and fluorescence emission spectra for FMN (green), FAD (black), DHOD B (red) and LOT6P (blue). All samples were dissolved on pH = 7.4 phosphate buffer. Right panels) solid lines: fluorescence (400 nm excitation) spectra, solid lines and symbols: two-photon excited (800 nm) fluorescence spectra. The absorption spectra were in good agreement with previous reports.5-6, 12, 23 The FMN concentration in LOT6P was calculated using ε450nm (FMN) = 12500 M-1 cm-1 and total flavin (FMN+FAD) concentration in DHOD B was calculated by means of ε450nm (FAD) = 11300 M-1 cm-1.42 The one-photon fluorescence emission spectra show the characteristic S1 → S0 emission band of flavins (FMN and FAD) and flavoproteins. These spectra do not show any shoulders and are symmetric to the S0 → S1 absorption band with a maximum at ~ 530 nm. The studied systems do not show linear absorption on the 800 nm region (Figure 2). We carried out the experiments to study the classical and entangled TPA process at 800 nm for accessing the low-lying excited states of FMN, FAD and two flavoproteins, DHOD B and LOT6PF. For flavins, the S1 excited state transition band has a ππ* (H → L) character with the highest oscillator strength. The S2 absorption band has a main contribution from a ππ* (H-1 → L) character transition (second highest oscillator strength) accompanied by minor contributions of nNπ* transitions.28, 43 Two-photon fluorescence spectra (Figure 2) closely followed the one-photon fluorescence spectra. The intensity of the maximum emission wavelength on the TPA induced fluorescence spectra (530 nm) was used for the power scans which allowed the calculation of the classical TPA cross-section at 800 nm, δTPA, see left part of Figure 3 and inset. Coumarin 153 in ethanol was used as a reference for the δTPA calculation, see details T. Goodson III 3 ACS Paragon Plus Environment


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on the supporting information. δTPA values for LOT6P, DHOD B, FMN and FAD are collected on Table 1.

Figure 3. Classical two-photon fluorescence power scan (left) and entangled two-photon absorption curve (right) for DHOD B and LOT6P. Inset: Log (Fluorescence) vs Log (power) plot used for classical TPA cross-section calculation. All samples were dissolved on a pH = 7.4 potassium phosphate buffer and a 1 cm pathlength quartz cuvette was used on the experiments. C (Flav) = C (FMN) + C (FAD) for DHOD B. Table 1. Entangled and classical TPA cross-section at 800 nm for free and protein embedded FAD and FMN. 1GM = 10-50 cm4 s photon-1 molecule-1. 𝜎𝜎𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 (x10-18) (cm2 molecule-1)

𝛿𝛿𝑇𝑇𝑇𝑇𝑇𝑇 (GM)

FMN

1.4

1.6

FMN or FAD in DHOD B

1.9

5.0

FMN in LOT6P

2.4

1.1

System FAD

0.29

2.1

ETPA curves were obtained by exciting the samples with a 800 nm central wavelength source of entangled photons generated by type II SPDC in a BBO (β-Barium Borate ) crystal,44 for this light source the entanglement time, TE = 63 fs (see supporting information) was estimated previously.35 ETPA cross-sections reported in Table 1 were estimated from the slope of the ETPA curve (Figure 3), m, the chromophore concentration, C (mol L-1), the cuvette pathlength, l (1.0 cm in our experiments) and required conversion factors, see supporting information. We can order these values as, σETPA (FAD) < σETPA (FMN) < σETPA (flavin in DHOD B) < σETPA (FMN in LOT6P). T. Goodson III


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The trend for the classical TPA cross-section is δTPA (FMN) ~ δTPA (FMN in LOT6P) < δTPA (FAD) < δTPA (flavin in DHOD B). δTPA for FMN and FAD are in reasonable agreement with reported values of ~0.96 and ~1.2 GM respectively.25, 27 Moreover, is clear that from FAD to FMN, σETPA increases significantly, σETPA (FMN) ~ 5 σETPA (FAD). Notably, the same is true for the 400 nm linear absorption, ε (FMN) > ε (FAD). In contrast, the classical TPA cross-section at 800 nm practically does not change for FMN and FAD. This difference between σETPA and δTPA of FMN and FAD can be explained as follows. First, it is important to mention that for flavins, exciting with 400 nm (one-photon absorption) or 800 nm (two-photon absorption) generates a superposition of the S1 and S2 excited states. This last means that 800 nm TPA has S0 → S1 and S0 → S2 contributions. In fact, for TPA or ETPA presented here do not correspond to pure electronic transitions, but to vibronic transitions to S1,ν ≥ 4 and S2,ν ≥ 2, see supporting information. Considering μjk the transition (j≠k) or permanent (j = k) dipole moment, δTPA scales as34: 𝛿𝛿𝑇𝑇𝑇𝑇𝑇𝑇 ∝ �

1

𝑖𝑖𝜅𝜅 �𝜔𝜔0 + 𝜀𝜀𝑔𝑔 − 𝜀𝜀𝑒𝑒 � − 2𝑒𝑒

𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒𝜇𝜇𝑒𝑒𝑒𝑒 ∙ 𝑒𝑒 +

1

𝑖𝑖𝜅𝜅𝑔𝑔 𝜔𝜔0 − 2

𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒𝜇𝜇𝑔𝑔𝑔𝑔 ∙ 𝑒𝑒 +

1

𝑖𝑖𝜅𝜅𝑓𝑓 −𝜔𝜔0 − 2

𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒�

2

(𝟏𝟏)

f, e and g represent final, intermediate and ground states, ω0 is the one-photon energy, εi is the energy of an electronic state i and κj is the linewidth of a transition to a j state. Likewise, the ETPA cross-section scales as34: 𝜎𝜎𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸

𝜅𝜅𝑒𝑒 𝑇𝑇𝑒𝑒

1 − 𝑒𝑒 −𝑖𝑖�𝜔𝜔0 +𝜀𝜀𝑔𝑔−𝜀𝜀𝑒𝑒�𝑇𝑇𝑒𝑒− 2 1 − 𝑒𝑒 −𝑖𝑖𝜔𝜔0 𝑇𝑇𝑒𝑒− ∝� 𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒𝜇𝜇𝑒𝑒𝑒𝑒 ∙ 𝑒𝑒 + 𝑖𝑖𝜅𝜅 𝑖𝑖𝜅𝜅𝑔𝑔 �𝜔𝜔0 + 𝜀𝜀𝑔𝑔 − 𝜀𝜀𝑒𝑒 � − 2𝑒𝑒 𝜔𝜔0 − 2 +

1 − 𝑒𝑒

𝑖𝑖𝜔𝜔0 𝑇𝑇𝑒𝑒 −

𝜅𝜅𝑓𝑓 𝑇𝑇𝑒𝑒 2

𝑖𝑖𝜅𝜅𝑓𝑓 −𝜔𝜔0 − 2

𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒�

2

𝜅𝜅𝑔𝑔 𝑇𝑇𝑒𝑒 2

𝜇𝜇𝑓𝑓𝑓𝑓 ∙ 𝑒𝑒𝜇𝜇𝑔𝑔𝑔𝑔 ∙ 𝑒𝑒

(𝟐𝟐)

Te is the entanglement time of the photon pair. It seems that the transition dipole moment contributions (𝜇𝜇𝑆𝑆1 𝑆𝑆0 ∙ 𝜇𝜇𝑆𝑆0 𝑆𝑆0 , 𝜇𝜇𝑆𝑆1 𝑆𝑆1 ∙ 𝜇𝜇𝑆𝑆1 𝑆𝑆0 , 𝜇𝜇𝑆𝑆2 𝑆𝑆0 ∙ 𝜇𝜇𝑆𝑆0 𝑆𝑆0 and 𝜇𝜇𝑆𝑆2 𝑆𝑆2 ∙ 𝜇𝜇𝑆𝑆2 𝑆𝑆0 ) have a distinctive effect on the ETPA process given that 𝜇𝜇𝑆𝑆1 𝑆𝑆0 and 𝜇𝜇𝑆𝑆2 𝑆𝑆0 must be higher in 2

FMN compared to FAD, εfg ∝ �𝜇𝜇𝑓𝑓𝑓𝑓 � and ε400nm (FMN) ~ 9375 M-1cm-1 > ε400nm (FAD) ~ 6639 M-1 cm-1. Furthermore, these transition dipole moment contributions must be stronger for the chromophore embedded in the protein than for the free chromophore. Related effects on the improvement of the linear absorption have been observed for FMN. The extinction coefficient at 450 nm has been reported to increase when FMN is non-covalently bound to iLOV protein,26 it varies from 12200 to ~ 17600 M-1 cm-1. For miniSOG,28 another FMN protein, it increases from 12063 M-1 cm-1 to 15833 M-1 cm-1. Conversely, for free FMN the δTPA at 800 nm is slightly higher (~ 0.6 GM) than FMN in the miniSOG protein. The same is true for iLOV. For LipDH (lipoamide dehydrogenase, FAD cofactor) δTPA at 800 nm is ~ 3.7 GM,27 this value is not excessively higher than the corresponding for FAD, ~ 1.2 GM. For DHOD B δTPA at 800 nm (5.0 GM) is higher than the respective quantities for FMN or FAD (see Table 1). These last comparisons suggest that at 800 nm, δTPA is not substantially affected for the flavins when the chromophore is embedded in the protein. However, in the case of ETPA the increase on σETPA is to a certain degree more marked than δTPA. For instance, a ~6.5x increase on δTPA might be feasible when going from free FAD to FAD embedded in DHOD B. To conclude, σETPA significantly increases for FAD compare to FMN, instead δTPA are virtually the same for FAD and FMN. We found that the ETPA for FMN and FAD increases on going from the free T. Goodson III 5 ACS Paragon Plus Environment


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chromophores in solution to the chromophores embedded in the protein. Additionally, σETPA at 800 nm estimated for the systems studied here are just one order of magnitude lower than the linear absorption cross-section at 400 nm, ~ 3.6x10-17 and 2.5x10-17 cm2 molecule-1 for FMN and FAD. The distinctive increase on σETPA for LOT6P or DHOD B compared with the free flavins, indicates that the unique properties of entangled photons contribute to a more clear manifestation of the enhancement on the TPA related properties, i.e. transition and permanent dipole moments, when the chromophores are embedded in the protein. Our findings settle the basis for the study of the ETPA and ETPA induced phenomena (charge transfer, proton transfer, Förster energy transfer and signaling) properties of many other reported fluorescent proteins with high TPA cross-section peaks at 800 nm1 and potentially high σETPA. 800 nm is a particularly convenient wavelength because entangled photons at that wavelength can be easily obtained using SPDC pumped by the second harmonic of conventional fs pulsed Ti:Sapphire lasers or even ~ 400 nm CW lasers. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Samples preparation, steady state spectroscopy, TPA experimental setup description, TPA curve for coumarin 153 and ETPA experimental setup description. AUTHOR INFORMATION Corresponding Author *[email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by Air Force Office of Scientific Research (Biophysics) Grant # FA9550-17-10457 (TGIII). Authors thank Dr. Sonja Sollner for LOT6P protein preparation and Dr. Zainab Bellow for DHOD B protein preparation. REFERENCES 1. Drobizhev, M.; Makarov, N. S.; Tillo, S. E.; Hughes, T. E.; Rebane, A., Nat. Methods 2011, 8, 393. 2. Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W., Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 10763. 3. Chen, Y.; Periasamy, A., Microsc. Res. Tech. 2003, 63, 72. 4. Liger, D.; Graille, M.; Zhou, C.-Z.; Leulliot, N.; Quevillon-Cheruel, S.; Blondeau, K.; Janin, J.; van Tilbeurgh, H., J. Biol. Chem. 2004, 279, 34890. 5. Sollner, S.; Nebauer, R.; Ehammer, H.; Prem, A.; Deller, S.; Palfey, B. A.; Daum, G.; Macheroux, P., FEBS J. 2007, 274, 1328. 6. Nielsen, F. S.; Andersen, P. S.; Jensen, K. F., J. Biol. Chem. 1996, 271, 29359. 7. Sollner, S.; Deller, S.; Macheroux, P.; Palfey, B. A., Biochemistry 2009, 48, 8636. 8. Javier, G.; Isabel, S.-V.; Jesús, N.; Susana de, M., Methods Appl. Fluores. 2016, 4, 042005. 9. Pan, J.; Byrdin, M.; Aubert, C.; Eker, A. P. M.; Brettel, K.; Vos, M. H., J. Phys. Chem. B 2004, 108, 10160. 10. Park, H. W.; Kim, S. T.; Sancar, A.; Deisenhofer, J., Science 1995, 268, 1866. 11. Cashmore, A. R.; Jarillo, J. A.; Wu, Y.-J.; Liu, D., Science 1999, 284, 760. 12. Islam, S. D. M.; Susdorf, T.; Penzkofer, A.; Hegemann, P., Chem. Phys. 2003, 295, 137. 13. Kao, Y.-T.; Saxena, C.; He, T.-F.; Guo, L.; Wang, L.; Sancar, A.; Zhong, D., J. Am. Chem. Soc. 2008, 130, 13132. 14. Mataga, N.; Chosrowjan, H.; Shibata, Y.; Tanaka, F., J. Phys. Chem. B 1998, 102, 7081. 15. Mataga, N.; Chosrowjan, H.; Taniguchi, S.; Tanaka, F.; Kido, N.; Kitamura, M., J. Phys. Chem. B 2002, 106, 8917. 16. Sengupta, A.; Khade, R. V.; Hazra, P., J. Photochem. Photobiol., A 2011, 221, 105. 17. Visser, A. J. W. G., Photochem. Photobiol. 1984, 40, 703.

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