No Photon Wasted: An Efficient and Selective Singlet Oxygen

Sep 11, 2017 - Additionally, the authors acknowledge the use of the FLIMfit software tools developed at Imperial College London and the UCSF Chimera p...
37 downloads 23 Views 1MB Size
Download PDF
Article pubs.acs.org/JPCB

No Photon Wasted: An Efficient and Selective Singlet Oxygen Photosensitizing Protein Michael Westberg,† Mikkel Bregnhøj,† Michael Etzerodt,‡ and Peter R. Ogilby*,† †

Department of Chemistry, Aarhus University, DK-8000 Aarhus, Denmark Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark

‡

S Supporting Information *

ABSTRACT: Optogenetics has been, and will continue to be, a boon to mechanistic studies of cellular processes. Genetically encodable proteins that sensitize the production of reactive oxygen species (ROS) are expected to play an increasingly important role, particularly in elucidating mechanisms of temporally and spatially dependent cell signaling. However, a substantial challenge in developing such photosensitizing proteins has been to funnel the optical excitation energy into the initial selective production of only one ROS. Singlet molecular oxygen, O2(a1Δg), is a ROS known to have a wide range of effects on cell function. Nevertheless, mechanistic details of singlet oxygen’s behavior in a cell are lacking. On the basis of the rational optimization of a LOV-derived flavoprotein, we now report the development and photophysical characterization of a protein-encased photosensitizer that efficiently and selectively produces singlet oxygen at the expense of other ROS, especially ROS that derive from photoinduced electron transfer reactions. These results set the stage for a plethora of new experiments to elucidate ROS-mediated events in cells.

■

INTRODUCTION The palette of genetically encodable tags that produce reactive oxygen species (ROS) upon light absorption is rapidly expanding, contributing important and exciting developments for the optogenetic toolbox.1−7 Such protein-based sensitizers have the highly desired properties of facilitating ROS production with targeted, molecularly defined spatial control via protein fusion and precise temporal and dose control through manipulation of the incident light.8,9 Consequently, optogenetic ROS sensitizers have already found extensive use as tags for chromophore-assisted light inactivation (CALI) of proteins and cells,7,10−12 correlated light and electron microscopy (CLEM),2,13 and mechanistic studies of the cellular effects of ROS,9,14,15 for example. All currently reported ROS photosensitizing proteins appear to produce the superoxide anion, O2•−, via photoinitiated electron transfer reactions to ground-state oxygen, O2(X3Σg−). In some cases, energy transfer from the excited-state sensitizer to O2(X3Σg−) kinetically competes with these reactions to also produce singlet oxygen, O2(a1Δg), the lowest excited state of molecular oxygen. However, to our knowledge, a genetically encodable photosensitizer that selectively produces O2(a1Δg) at the expense of O2•− has yet to be developed. The superoxide anion is relatively unreactive and only directly reacts at a few specific protein sites.16−18 In contrast, O2(a1Δg) reacts directly and efficiently with five common and functionally important amino acids (Cys, His, Met, Trp, Tyr).18,19 This fact, combined with the relatively limited diffusion distance of O2(a1Δg) in a cell (∼150 nm),8 indicates the unique potential of O2(a1Δg) photosensitizing proteins as a © 2017 American Chemical Society

way to elicit a localized perturbation of a cell. Indeed, the importance of such proteins is underscored by the ability of O2(a1Δg) to initiate a wide range of different cell responses that depend on both its subcellular site of formation and dose.8,20−22 However, a great deal remains to be learned about the chemical biology of O2(a1Δg), particularly its mechanisms of action at a molecular level. In this regard, one must ideally control the reactive intermediates involved when studying molecular mechanisms. Thus, an efficient and selective optogenetic O2(a1Δg) sensitizer is highly desired. Photosensitization of O2(a1Δg) is commonly achieved through energy transfer from the triplet state of the sensitizer to O2(X3Σg−).23,24 In this regard, flavoproteins appear as a good starting scaffold for the design of an optogenetic O2(a1Δg) sensitizer because the intersystem crossing rate to the flavin triplet state is generally large.25 Moreover, flavin mononucleotide, FMN, itself can produce O2(a1Δg) with a high quantum yield when freely dissolved in aqueous solution (ΦΔ ≈ 0.65).4,26 Designing efficient O2(a1Δg) generating flavoproteins has, however, turned out to be a challenging task. For example, the much publicized FMN-containing system called miniSOG (for mini singlet oxygen generator) has been shown to only produce O2(a1Δg) in low yields (ΦΔ ≈ 0.03).27,28 Our studies have shown that the efficiency of O2(a1Δg) sensitization by such LOV-derived flavoproteins is limited by the fraction of FMN triplet states, 3FMN, that are quenched by O2(X3Σg−).4,29 This is due to (1) inefficient O2(X3Σg−) Received: August 7, 2017 Published: September 11, 2017 9366

DOI: 10.1021/acs.jpcb.7b07831 J. Phys. Chem. B 2017, 121, 9366−9371

The Journal of Physical Chemistry B

■

diffusion through the protein scaffold and (2) quenching by competing undesired oxygen-independent pathways, including electron transfer to 3FMN from surrounding amino acids. Utilizing this knowledge, we constructed an improved Q103L30 mutant of miniSOG named SOPP (for singlet oxygen photosensitizing protein), for which ΦΔ ≈ 0.25.4 This larger quantum yield has since been confirmed by others,31 and in vivo experiments indicate that SOPP is indeed more efficient at initiating cell death than miniSOG.12 In developing SOPP, we focused on reducing the electron affinity of 3FMN by removing intermolecular H-bonds between FMN and the protein scaffold.4 We specifically removed the Hbond from Q103 to CO(4) in FMN without notably affecting the binding of FMN by the protein. Although this mutation increased ΦΔ, it did not completely preclude the photoinduced electron transfer reactions that lead to the production of O2•−. We now show that it is possible to appreciably increase both the yield and initial selectivity of O2(a1Δg) production by further manipulating the SOPP protein scaffold. Specifically, we show that using site-directed mutagenesis we can (1) tune the rate of 3FMN quenching by O2(X3Σg−) and (2) preclude electron transfer from the protein to 3FMN. To this end, we identified and removed the amino acid functionalities that participate in electron transfer to 3FMN through a targeted screen of the six potential electron-donating residues in the LOV core of SOPP (Figure 1).32 Additionally, we investigated

Article

RESULTS AND DISCUSSION

Initial Screening of SOPP Mutants. We employed sitedirected mutagenesis to target amino acids in SOPP that are potentially involved in either (1) electron transfer to 3FMN or (2) modulating oxygen diffusion through the protein (Figure 1). The resulting mutants were screened via two optical techniques that we have previously used to characterize the photophysics of 3FMN in both miniSOG and SOPP: transient absorption and O2(a1Δg) phosphorescence (Figure 2).4,29

Figure 1. Illustration of SOPP and the amino acids specifically targeted for site-directed mutagenesis: (green) FMN, (orange) potential electron-donating residues and targets for O2(a1Δg) reactions, and (blue) residue important for the electron affinity of 3FMN and oxygen diffusion into the active site. The illustration is based on the crystal structure of iLOV (PDB ID: 4EET, sequence identity 95%).35

Figure 2. Data recorded upon excitation of the SOPP W81L/L103V mutant (i.e., SOPP2). (top) Representative examples of the transient absorption traces recorded at 710 ± 5 nm and assigned to 3FMN. The data were fitted to a monoexponential decay function (black lines). (bottom) Representative time-resolved 1275 nm O2(a1Δg) phosphorescence trace. Details regarding the fit to the data (black line) and calculation of ΦΔ are presented in the SI. To capitalize on the H/D solvent isotope effect on the O2(a1Δg) lifetime,23 the data were recorded from D2O-based PBS solutions at 23 °C.

the effect of residue 103 on oxygen diffusion to encapsulated FMN; our independent studies on SOPP and miniSOG, as well as molecular dynamics simulations, have indicated that this residue might influence the accessibility of the chromophore to oxygen.29,33,34 In this way, we developed highly efficient O2(a1Δg) photosensitizing proteins, which we call SOPP2 and SOPP3. In SOPP3, (1) the O2(a1Δg) quantum yield, ΦΔ ≈ 0.60, is comparable to that of free FMN and (2) all photons absorbed translate solely into fluorescence or O2(a1Δg) production, indicating that we have indeed precluded the photoinitiated electron transfer reactions that result in the production of O2•−.

Transient absorption signals recorded at 710 nm were used to determine the lifetime of 3FMN, τT, under both air and oxygen-saturated conditions. Assuming that the rate of oxygenmediated quenching of 3FMN is linear in [O2(X3Σg−)], we are thus able to extract rate constants for both the oxygendependent and oxygen-independent deactivation channels (τT−1 = k0 + kq [O2(X3Σg−)]). On the basis of these parameters alone, an improved version of SOPP would show (1) a decrease in the magnitude of the oxygen-independent rate constant, k0, that characterizes both electron transfer to 3FMN and intersystem crossing to the FMN ground state, and/or (2) an increase in the bimolecular rate constant for O2(X3Σg−)mediated quenching of 3FMN, kq. To complement these data, we also determined values of ΦΔ using the flavoprotein9367

DOI: 10.1021/acs.jpcb.7b07831 J. Phys. Chem. B 2017, 121, 9366−9371

Article

The Journal of Physical Chemistry B photosensitized time-resolved O2(a1Δg) phosphorescence signal at 1275 nm. We first identified the electron-donating amino acids in SOPP by performing an Ala-screen of all six potential candidates (i.e., these residues were successively replaced with Ala). Mutation of the Y30 and Y73 residues, which are highly conserved in wild-type LOV proteins and their fluorescent derivatives,25,36,37 lead to loss of FMN binding by the proteins. FMN remains bound in the other four mutants (W81A, H85A, M89A, Y98A). When examining the photophysical data for these four mutants, the W81A mutant stands out; upon removal of this Trp residue and its associated indole ring, a significant reduction in k0 and a concomitant increase in ΦΔ are observed (Table 1). On the other hand, for the other three

Our second approach for optimizing SOPP focused on increasing the rate of oxygen diffusion through the protein, thereby increasing the magnitude of kq. We chose to focus on the L103 residue in SOPP because our independent investigations of SOPP and miniSOG indicated that this residue modulates kq.29 First, we mutated L103 into other aliphatic residues (Ala, Val, and Ile) that should (1) interact favorably with oxygen and (2) differentially facilitate the formation of the dynamic free volume necessary for oxygen diffusion.41−43 In addition, we also mutated L103 into alcohol containing amino acids (Ser and Thr) to explore the possibility for subtle structural rearrangement of the protein upon water coordination (e.g., formation of a water channel). Incorporation of the alcohol-containing amino acids led to an appreciable increase in k0 while not affecting kq, the combination of which resulted in a lower ΦΔ (Table 1). Among the aliphatic substitutions, the L103V mutant is particularly interesting because an appreciable increase in both kq and ΦΔ is observed. For this mutant, our value of ΦΔ is consistent with that obtained in an independent study.31 However, in this latter study, 3FMN lifetimes were not reported, and hence, an explanation for this increase in ΦΔ could not be provided.31 Our present data indicate that the positive effect of the L103V mutation on ΦΔ is correlated with more efficient oxygen diffusion through the protein. To determine if the improvements described above are additive, we created a series of mutants where the Trp residue at position 81 had been removed and the aliphatic side chain on the residue at position 103 varied (Table 1). Upon examining the photophysical data, the effects of the individual mutations indeed appear to be additive. For example, the W81L/L103V mutant has, relative to SOPP, both a smaller value of k0 and a larger value of kq. This particular combination results in a value of ΦΔ that is close to our previously estimated quantum yields of 3FMN formation in SOPP and miniSOG (ΦT ≈ 0.6).29 The W81A/L103V mutant has similar properties as the W81L/ L103V mutant. However, comparison of the thermally induced denaturation curves for these two proteins indicate that the complex between FMN and W81L/L103V is more stable (Figure S4). Consequently, we investigated the properties of the W81L/L103V mutant further. We now call this mutant SOPP2. Detailed Characterization of SOPP2. To further characterize the performance of SOPP2, we recorded SOPP2sensitized O2(a1Δg) phosphorescence signals and retrieved the associated values of ΦΔ and τT under a wide range of conditions (Figure S3 and Tables 2 and 3). When recording and analyzing these data, additional control experiments were

Table 1. Photophysical Properties Obtained in the Initial Screening of SOPP Mutantsa k0 [104 s−1]b

kq [107 M−1 s−1]c

ΦΔ (air)d

e

miniSOG SOPPe

2.41 0.51

0.56 1.02

0.03 0.23

SOPP SOPP SOPP SOPP SOPP

W81A W81L H85A M89A Y98A

0.15 0.13 0.75 0.66 0.57

0.63 0.77 0.78 0.86 0.91

0.55 0.54 0.25 0.24 0.24

SOPP SOPP SOPP SOPP SOPP

L103A L103I L103V L103S L103T

0.77 0.76 0.52 1.86 1.18

1.08 0.89 1.88 0.87 1.02

0.25 0.21 0.36 0.08 0.14

SOPP SOPP SOPP SOPP SOPP SOPP

W81A/L103A W81A/L103I W81A/L103V W81L/L103A W81L/L103I W81L/L103Vf

0.14 0.14 0.17 0.16 0.15 0.21

1.23 0.98 1.68 1.07 0.95 1.67

0.53 0.55 0.61 0.53 0.54 0.57

protein

Measurements were performed in D2O-based PBS buffer at 23 °C. Additional data are tabulated in Table S1. bEstimated precision of ±0.1 × 104 s−1. cEstimated precision of ±0.2 × 107 M−1 s−1. d Estimated precision of ±10%. eFrom Westberg et al.29 fSOPP2. Note that the numbers reported for SOPP2 in this table differ slightly from those reported in Tables 2 and 3. In the latter, a more comprehensive and accurate experimental procedure was used to obtain the data. A detailed discussion of these different experimental and analytical approaches is provided in the SI. a

Table 2. Quantum Yields of O2(a1Δg) Formation at Different Temperatures and Oxygen Concentrationsa

mutants, these photophysical parameters are very similar to those observed in SOPP. This suggests that W81 is the primary electron donor to 3FMN in SOPP. This observation stands in stark contrast to the report by Ruiz-González et al.,28 where no improvement of ΦΔ was found for the analogous W81F mutant of miniSOG. To help confirm our observations, we also purified and examined W81L and W81F mutants of SOPP. While the W81F mutation does not bind FMN, the W81L protein binds FMN and behaves similarly to the W81A mutant. On this basis, the Trp residue at position 81 appears to be the most efficient electron donor in SOPP. This conclusion is consistent with the fact that the pertinent Trp radical ion has been observed in a series of studies on nonadduct-forming LOV proteins.38−40

ΦΔ(23 °C)

ΦΔ(37 °C)

protein

5% O2

21% O2

100% O2

5% O2

21% O2

100% O2

miniSOGb SOPPb SOPP2c SOPP3c

0.01 0.07 0.29 0.50

0.03 0.23 0.51 0.61

0.14 0.44 0.55 0.60

0.01 0.09 0.28 0.50

0.04 0.27 0.50 0.59

0.20 0.48 0.58 0.61

a

Determined for proteins dissolved in D2O-based PBS buffer. bFrom Westberg et al.;29 the values at 5% O2 and 100% O2 were calculated using ΦΔ = (1 − Φf) · f T. cThese values are retrieved from the O2(a1Δg) traces shown in Figure S3. The estimated accuracy is ±10%. 9368

DOI: 10.1021/acs.jpcb.7b07831 J. Phys. Chem. B 2017, 121, 9366−9371

Article

The Journal of Physical Chemistry B Table 3. Deactivation Rates of Protein-Encapsulated 3FMNa 23 °C

37 °C

protein

k0 [104 s−1]d

kq [107 M−1 s−1]e

k0 [104 s−1]f

kq [107 M−1 s−1]g

miniSOGb SOPPb SOPP2c SOPP3c

2.41 0.51 0.10 0.03

0.56 1.02 2.20 2.33

2.96 0.76 0.17 0.01

1.38 2.41 4.80 5.35

a Determined for proteins dissolved in D2O-based PBS buffer. bFrom Westberg et al.29 cThese values are based on the 3FMN lifetimes retrieved from fits to the O2(a1Δg) traces shown in Figure S3. dEstimated accuracy of ±0.08 × 104 s−1. eEstimated accuracy of ±0.2 × 107 M−1 s−1. fEstimated accuracy of ±0.14 × 104 s−1. gEstimated accuracy of ±0.4 × 107 M−1 s−1.

Table 4. Singlet-State Photophysical Propertiesa

performed to ensure the accuracy of the reported ΦΔ under all conditions (see the SI for details). We first focus on the data recorded from air-saturated solutions at 23 °C. In our independent work on SOPP and miniSOG,29 we showed that the quantum yields of 3FMN, ΦT, were related to the fluorescence quantum yield, Φf, through the expression ΦT = 1 − Φf. In other words, the excited singlet state of protein-encapsulated FMN is deactivated mainly via fluorescence and intersystem crossing to 3FMN. For SOPP2, we find Φf = 0.41 ± 0.02. When combined with the ΦΔ value of 0.51 ± 0.05 (Table 2) for this same mutant, the sum ΦΔ + Φf approaches 1. Moreover, the O2(a1Δg) quantum yield of SOPP2 does not increase significantly upon saturating the solution with oxygen (ΦΔ = 0.55 ± 0.06), indicating that nearly all 3FMN states formed are quenched by O2(X3Σg−) in the airsaturated system. Thus, for SOPP2 in D2O-based PBS buffer at 23 °C, electron transfer reactions involving 3FMN do not efficiently compete with O2(a1Δg) production. To investigate the performance of SOPP2 under conditions that may be more relevant for experiments performed in cells, we also determined O2(a1Δg) quantum yields in solutions at 37 °C and/or equilibrated with an atmosphere containing only 5% O2 (Table 2). Although a reduction in ΦΔ is indeed expected when the partial pressure of oxygen in the sample is reduced, SOPP2 nevertheless retains a large ΦΔ, in stark contrast to miniSOG and SOPP. However, it is also apparent that ΦΔ for SOPP2 is no longer close to the desired limit of ∼0.6 at this lower oxygen concentration. Additional Mutations to Generate SOPP3. In an attempt to further increase values of ΦΔ, particularly under conditions of low oxygen concentration, we performed another round of mutagenesis starting with SOPP2. Specifically, with the removal of W81, other residues, in turn, may become competitive electron donors. Thus, the rationale was to mutate H85, M89, and Y98 into residues that would not only be improbable electron donors32 but that would also be poorer substrates for O2(a1Δ g) oxidation and the subsequent formation of downstream ROS (e.g., formation and decomposition of hydroperoxides).19 The latter phenomenon would clearly mitigate the mechanistic advantage accrued with an increase in ΦΔ. We chose to focus on these specific residues because our initial Ala-screen indicated that the protein scaffold is relatively stable toward changes at these positions (vide supra). With inspiration from a range of wild-type LOV proteins and their fluorescent derivatives,25,36,37 we screened seven new mutants (SI). On the basis of this screening, we chose the W81L/H85N/M89I/Y98A/L103V mutant for further characterization. We now call this new mutant SOPP3. The singlet-state properties and thermal stability of SOPP2 and SOPP3 are almost identical (Table 4 and Figure S4). More strikingly, SOPP3 generates O2(a1Δg) with enhanced efficiency

protein c

miniSOG SOPPc SOPP2 SOPP3

λabsmax [nm]d

εmaxb [mM−1 cm−1]

λflumax [nm]e

τs [ns]f

Φfg

447 439 439 439

14.1 14.5 14.8 15.0

501/526 488/515 491/513 490/514

4.96 4.19 4.45 4.34

0.43 0.39 0.41 0.41

Measured in D2O-based PBS buffer at 23 °C, unless otherwise noted. Additional data recorded in H2O-based PBS buffer and/or at 37 °C are tabulated in Table S4. bDetermined in H2O-based PBS buffer with an estimated accuracy of ±0.3 mM−1 cm−1. cFrom Westberg et al.4,29 d Estimated accuracy of ±1 nm. eEstimated accuracy of ±2 nm. f Estimated accuracy of ±0.15 ns. gEstimated accuracy of ±0.02. a

under all conditions examined. In particular, we note an increase in ΦΔ at low oxygen concentrations relative to SOPP2 (Table 2) concomitant with systematic changes in τT (Figure S3), which indicates that the beneficial effects of this second round of mutations mainly arise from an additional decrease in k0 (Table 3). Thus, with SOPP3, it appears that we have now reached the desired limit of ΦΔ + Φf = 1 for a range of conditions relevant for studies with cells. At this limit, every photon absorbed by SOPP3 is constructively spent either on fluorescence (e.g., useful for imaging of tag localization) or energy transfer to form O2(a1Δg). Blue- vs Red-Light-Absorbing Optogenetic Proteins. Flavin-based optogenetic tags or fluorescent proteins absorb light at the blue end of the visible spectrum (Figure 3). Thus, when employed in live cells, the incident light could also excite endogenous chromophores in these cells. Specifically, when

Figure 3. Normalized absorption and fluorescence spectra of miniSOG, SOPP, and SOPP3 in D2O-based PBS at 23 °C. Spectra recorded in H2O-based PBS are identical. Also indicated is the 514 nm laser line often used to excite fluorescent probes during cell imaging. 9369

DOI: 10.1021/acs.jpcb.7b07831 J. Phys. Chem. B 2017, 121, 9366−9371

Article

The Journal of Physical Chemistry B using miniSOG, SOPP, or SOPP3, endogenous flavoproteins and free intracellular flavins will certainly also be excited, which, in the least, will hamper attempts to spatially control the O2(a1Δg) dose. However, these effects may be mitigated when using SOPP3 instead of SOPP or miniSOG because a smaller light dose should be needed to create the same amount of O2(a1Δg). As an alternative to blue-light-absorbing optogenetic tags that generate ROS, we must mention a recently developed red-lightabsorbing system in which an iodine-substituted variant of malachite green is used as the sensitizer.6 Advantages of this system include the following: (a) the malachite green analogue is an exogenous chromophore that is benign as an O2(a1Δg) sensitizer unless bound in the protein, and (b) there is little inherent competition from endogenous chromophores for the incident light. However, in comparison to SOPP3, this malachite-green-derived system does not make O2(a1Δg) in great yield (ΦΔ ≈ 0.13), and the initial selectivity of this system with respect to the production of O2(a1Δg) has yet to be demonstrated. An often-overlooked advantage of blue-light-absorbing optogenetic systems is that they can be simultaneously used with fluorescent tags that have red-shifted absorption spectra. This has a pronounced advantage if, for example, an experiment requires just a short perturbation of the cell via blue-light excitation of the sensitizer (e.g., studies of ROS stimulated cell proliferation22), while the subsequent long-term observation of cell response can be done using a red-light-absorbing fluorescent probe. Specifically, note that the SOPP family can be used with popular fluorescent probes that can be excited at λ > 500 nm (Figure 3).44,45 In contrast, optogenetic ROS sensitizers that have their main absorption band in the red or near-infrared generally also have absorption bands at shorter wavelengths. Therefore, such systems will not be completely compatible with the many blue/green-light-absorbing fluorophores commonly used to probe cell function (i.e., additional and undesired ROS may be created by the sensitizer during the process of monitoring cell response to an initial controlled dose of ROS). In any event, the development of a palette of efficient ROS photosensitizing proteins with different absorption spectra is important because different applications will invariably have different requirements.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Peter R. Ogilby: 0000-0003-0165-5159 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by a grant from the Danish National Research Foundation. The authors thank Professor Daniel E. Otzen (Aarhus University) for access to the CD spectrometer and Anette Kjems (Aarhus University) for assistance with protein mutation, expression, and purification. Additionally, the authors acknowledge the use of the FLIMfit software tools developed at Imperial College London and the UCSF Chimera package from the Computer Graphics Laboratory, University of California San Francisco.

■

REFERENCES

(1) Serebrovskaya, E. O.; Edelweiss, E. F.; Stremovskiy, O. A.; Lukyanov, K. A.; Chudakov, D. M.; Deyev, S. M. Targeting Cancer Cells by Using an Antireceptor Antibody-Photosensitizer Fusion Protein. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 9221−9225. (2) Shu, X.; Lev-Ram, V.; Deerinck, T. J.; Qi, Y.; Ramko, E. B.; Davidson, M. W.; Jin, Y.; Ellisman, M. H.; Tsien, R. Y. A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms. PLoS Biol. 2011, 9, e1001041. (3) Torra, J.; Burgos-Caminal, A.; Endres, S.; Wingen, M.; Drepper, T.; Gensch, T.; Ruiz-González, R.; Nonell, S. Singlet Oxygen Photosensitisation by the Fluorescent Protein Pp2FbFP L30M, a Novel Derivative of Pseudomonas Putida Flavin-Binding Pp2FbFP. Photochem. Photobiol. Sci. 2015, 14, 280−287. (4) Westberg, M.; Holmegaard, L.; Pimenta, F. M.; Etzerodt, M.; Ogilby, P. R. Rational Design of an Efficient, Genetically Encodable, Protein-Encased Singlet Oxygen Photosensitizer. J. Am. Chem. Soc. 2015, 137, 1632−1642. (5) Sarkisyan, K. S.; Zlobovskaya, O. A.; Gorbachev, D. A.; Bozhanova, N. G.; Sharonov, G. V.; Staroverov, D. B.; Egorov, E. S.; Ryabova, A. V.; Solntsev, K. M.; Mishin, A. S.; et al. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PLoS One 2015, 10, e0145287. (6) He, J.; Wang, Y.; Missinato, M. A.; Onuoha, E.; Perkins, L. A.; Watkins, S. C.; St Croix, C. M.; Tsang, M.; Bruchez, M. P. A Genetically Targetable near-Infrared Photosensitizer. Nat. Methods 2016, 13, 263−268. (7) Makhijani, K.; To, T.-L.; Ruiz-González, R.; Lafaye, C.; Royant, A.; Shu, X. Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System. Cell Chem. Biol. 2017, 24, 110−119. (8) Westberg, M.; Bregnhøj, M.; Blázquez-Castro, A.; Breitenbach, T.; Etzerodt, M.; Ogilby, P. R. Control of Singlet Oxygen Production in Experiments Performed on Single Mammalian Cells. J. Photochem. Photobiol., A 2016, 321, 297−308. (9) Wojtovich, A. P.; Foster, T. H. Optogenetic Control of ROS Production. Redox Biol. 2014, 2, 368−376. (10) Lin, J. Y.; Sann, S. B.; Zhou, K.; Nabavi, S.; Proulx, C. D.; Malinow, R.; Jin, Y.; Tsien, R. Y. Optogenetic Inhibition of Synaptic Release with Chromophore-Assisted Light Inactivation (CALI). Neuron 2013, 79, 241−253. (11) Wojtovich, A. P.; Wei, A. Y.; Sherman, T. A.; Foster, T. H.; Nehrke, K. Chromophore-Assited Light Inactivation of Mitochondrial

■

CONCLUSIONS Through rational and systematic optimization of a LOV-derived flavoprotein, we identified and mutated key residues to yield a system in which (1) the rate constant for 3FMN quenching by O2(X3Σg−) is comparatively large and (2) photoinitiated electron transfer reactions from the protein to 3FMN have been precluded. In this report, we have characterized the fundamental photophysics of what we believe is the first selective and highly efficient genetically encodable O2(a1Δg) sensitizer. This system has the potential to become a valuable addition to the optogenetic toolbox.

■

additional data examples, and temperature stability of selected mutants (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b07831. Materials and methods, data compilations for both the first and second screening of the SOPP mutants, 9370

DOI: 10.1021/acs.jpcb.7b07831 J. Phys. Chem. B 2017, 121, 9366−9371

Article

The Journal of Physical Chemistry B Electron Transport Chain Complex II in Caenorhabditis elegans. Sci. Rep. 2016, 6, 29695. (12) Xu, S.; Chisholm, A. D. Highly Efficient Optogenetic Cell Ablation in C. elegans Using Membrane-Targeted miniSOG. Sci. Rep. 2016, 6, 21271. (13) Boassa, D.; Berlanga, M. L.; Yang, M. A.; Terada, M.; Hu, J.; Bushong, E. A.; Hwang, M.; Masliah, E.; George, J. M.; Ellisman, M. H. Mapping the Subcellular Distribution of α-Synuclein in Neurons Using Genetically Encoded Probes for Correlated Light and Electron Microscopy: Implications for Parkinson’s Disease Pathogenesis. J. Neurosci. 2013, 33, 2605−2615. (14) Ryumina, A. P.; Serebrovskaya, E. O.; Shirmanova, M. V.; Snopova, L. B.; Kuznetsova, M. M.; Turchin, I. V.; Ignatova, N. I.; Klementieva, N. V.; Fradkov, A. F.; Shakhov, B. E.; et al. Flavoprotein miniSOG as a Genetically Encoded Photosensitizer for Cancer Cells. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 5059−5067. (15) Noma, K.; Jin, Y. Optogenetic Mutagenesis in Caenorhabditis Elegans. Nat. Commun. 2015, 6, 8868. (16) D’Autréaux, B.; Toledano, M. B. ROS as Signalling Molecules: Mechanisms That Generate Specificity in ROS Homeostasis. Nat. Rev. Mol. Cell Biol. 2007, 8, 813−824. (17) Winterbourn, C. C. Reconciling the Chemistry and Biology of Reactive Oxygen Species. Nat. Chem. Biol. 2008, 4, 278−286. (18) Davies, M. J. Protein Oxidation and Peroxidation. Biochem. J. 2016, 473, 805−825. (19) Davies, M. J. Singlet Oxygen-Mediated Damage to Proteins and Its Consequences. Biochem. Biophys. Res. Commun. 2003, 305, 761− 770. (20) Redmond, R. W.; Kochevar, I. E. Spatially Resolved Cellular Responses to Singlet Oxygen. Photochem. Photobiol. 2006, 82, 1178− 1186. (21) Klotz, L.-O.; Kröncke, K.-D.; Sies, H. Singlet Oxygen-Induced Signaling Effects in Mammalian Cells. Photochem. Photobiol. Sci. 2003, 2, 88−94. (22) Blázquez-Castro, A.; Breitenbach, T.; Ogilby, P. R. Singlet Oxygen and ROS in a New Light: Low-Dose Subcellular Photodynamic Treatment Enhances Proliferation at the Single Cell Level. Photochem. Photobiol. Sci. 2014, 13, 1235−1240. (23) Ogilby, P. R. Singlet Oxygen: There Is Indeed Something New under the Sun. Chem. Soc. Rev. 2010, 39, 3181−3209. (24) Schweitzer, C.; Schmidt, R. Physical Mechanisms of Generation and Deactivation of Singlet Oxygen. Chem. Rev. 2003, 103, 1685− 1757. (25) Losi, A.; Gärtner, W. Solving Blue-Light Riddles: New Lessons from Flavin-Binding LOV Photoreceptors. Photochem. Photobiol. 2017, 93, 141−158. (26) Baier, J.; Maisch, T.; Maier, M.; Engel, E.; Landthaler, M.; Bäumler, W. Singlet Oxygen Generation by UVA Light Exposure of Endogenous Photosensitizers. Biophys. J. 2006, 91, 1452−1459. (27) Pimenta, F. M.; Jensen, R. L.; Breitenbach, T.; Etzerodt, M.; Ogilby, P. R. Oxygen-Dependent Photochemistry and Photophysics of “MiniSOG,” a Protein-Encased Flavin. Photochem. Photobiol. 2013, 89, 1116−1126. (28) Ruiz-González, R.; Cortajarena, A. L.; Mejias, S. H.; Agut, M.; Nonell, S.; Flors, C. Singlet Oxygen Generation by the Genetically Encoded Tag miniSOG. J. Am. Chem. Soc. 2013, 135, 9564−9567. (29) Westberg, M.; Bregnhøj, M.; Etzerodt, M.; Ogilby, P. R. Temperature Sensitive Singlet Oxygen Photosensitization by LOVDerived Fluorescent Flavoproteins. J. Phys. Chem. B 2017, 121, 2561− 2574. (30) In this paper, we use the numbering system of residues used in the original miniSOG report (Shu et al.2), which differs from the numbering system that we used in our first publication on SOPP (Westberg et al.4). (31) Rodríguez-Pulido, A.; Cortajarena, A. L.; Torra, J.; RuizGonzález, R.; Nonell, S.; Flors, C. Assessing the Potential of Photosensitizing Flavoproteins as Tags for Correlative Microscopy. Chem. Commun. 2016, 52, 8405−8408.

(32) Heelis, P. F.; Parsons, B. J.; Phillips, G. O.; McKellar, J. F. A Laser Flash Photolysis Study of the Nature of Flavin Mononucleotide Triplet States and the Reactions of the Neutral Form with Amino Acids. Photochem. Photobiol. 1978, 28, 169−173. (33) Pietra, F. Molecular Dynamics Simulation of Dioxygen Pathways through Mini Singlet Oxygen Generator (miniSOG), a Genetically Encoded Marker and Killer Protein. Chem. Biodiversity 2014, 11, 1883−1891. (34) Song, S.-H.; Freddolino, P. L.; Nash, A. I.; Carroll, E. C.; Schulten, K.; Gardner, K. H.; Larsen, D. S. Modulating LOV Domain Photodynamics with a Residue Alteration Outside the Chromophore Binding Site. Biochemistry 2011, 50, 2411−2423. (35) Christie, J. M.; Hitomi, K.; Arvai, A. S.; Hartfield, K. A.; Mettlen, M.; Pratt, A. J.; Tainer, J. A.; Getzoff, E. D. Structural Tuning of the Fluorescent Protein iLOV for Improved Photostability. J. Biol. Chem. 2012, 287, 22295−22304. (36) Wingen, M.; Potzkei, J.; Endres, S.; Casini, G.; Rupprecht, C.; Fahlke, C.; Krauss, U.; Jaeger, K.-E.; Drepper, T.; Gensch, T. The Photophysics of LOV-Based Fluorescent Proteins - New Tools for Cell Biology. Photochem. Photobiol. Sci. 2014, 13, 875−883. (37) Mukherjee, A.; Weyant, K. B.; Agrawal, U.; Walker, J.; Cann, I. K. O.; Schroeder, C. M. Engineering and Characterization of New LOV-Based Fluorescent Proteins from Chlamydomonas Reinhardtii and Vaucheria Frigida. ACS Synth. Biol. 2015, 4, 371−377. (38) Eisenreich, W.; Joshi, M.; Weber, S.; Bacher, A.; Fischer, M. Natural Abundance Solution 13C NMR Studies of a Phototropin with Photoinduced Polarization. J. Am. Chem. Soc. 2008, 130, 13544− 13545. (39) Eisenreich, W.; Fischer, M.; Römisch-Margl, W.; Joshi, M.; Richter, G.; Bacher, A.; Weber, S. Tryptophan 13C Nuclear-Spin Polarization Generated by Intraprotein Electron Transfer in a LOV2 Domain of the Blue-Light Receptor Phototropin. Biochem. Soc. Trans. 2009, 37, 382−386. (40) Thamarath, S. S.; Heberle, J.; Hore, P. J.; Kottke, T.; Matysik, J. Solid-State Photo-CIDNP Effect Observed in Phototropin LOV1C57S by 13C Magic-Angle Spinning NMR Spectroscopy. J. Am. Chem. Soc. 2010, 132, 15542−15543. (41) Cohen, J.; Schulten, K. O2 Migration Pathways Are Not Conserved across Proteins of a Similar Fold. Biophys. J. 2007, 93, 3591−3600. (42) Leroux, F.; Dementin, S.; Burlat, B.; Cournac, L.; Volbeda, A.; Champ, S.; Martin, L.; Guigliarelli, B.; Bertrand, P.; Fontecilla-Camps, J.; et al. Experimental Approaches to Kinetics of Gas Diffusion in Hydrogenase. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 11188−11193. (43) Baron, R.; Riley, C.; Chenprakhon, P.; Thotsaporn, K.; Winter, R. T.; Alfieri, A.; Forneris, F.; van Berkel, W. J. H.; Chaiyen, P.; Fraaije, M. W.; et al. Multiple Pathways Guide Oxygen Diffusion into Flavoenzyme Active Sites. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 10603−10608. (44) Rodriguez, E. A.; Campbell, R. E.; Lin, J. Y.; Lin, M. Z.; Miyawaki, A.; Palmer, A. E.; Shu, X.; Zhang, J.; Tsien, R. Y. The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem. Sci. 2017, 42, 111−129. (45) Cranfill, P. J.; Sell, B. R.; Baird, M. A.; Allen, J. R.; Lavagnino, Z.; de Gruiter, H. M.; Kremers, G.-J.; Davidson, M. W.; Ustione, A.; Piston, D. W. Quantitative Assessment of Fluorescent Proteins. Nat. Methods 2016, 13, 557−562.

9371

DOI: 10.1021/acs.jpcb.7b07831 J. Phys. Chem. B 2017, 121, 9366−9371