NONLINEAR OPTICAL PROPERTIES OF

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1. Introduction. Just like any other protein, green fluorescent protein (GFP) consists of an .... latter reasons make scientists more interested in using the more red-shifted FPs in single-photon ..... from Discosoma sp. red fluorescent protein. Nat.
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Journal of Nonlinear Optical Physics & Materials Vol. 19, No. 1 (2010) 1–13 c World Scientific Publishing Company  DOI: 10.1142/S0218863510005054

NONLINEAR OPTICAL PROPERTIES OF mSTRAWBERRY AND mCHERRY FOR SECOND HARMONIC IMAGING

EVELIEN DE MEULENAERE∗,†,‡,¶ , MARC DE WERGIFOSSE§ , EDITH BOTEK§ , STIJN SPAEPEN∗ , BENOˆIT CHAMPAGNE§, , JOS VANDERLEYDEN∗,‡ and KOEN CLAYS†,‡,∗∗ ∗Centre

of Microbial and Plant Genetics, University of Leuven, Kasteelpark Arenberg 20, BE-3001 Leuven, Belgium †Department

of Chemistry, University of Leuven, Celestijnenlaan 200D, BE-3001 Leuven, Belgium

‡Institute

for Nanoscale Physics and Chemistry (INPAC), University of Leuven, Belgium

§Laboratoire de Chimie Th´ eorique (LCT), Facult´ es Universitaires Notre-Dame de la Paix (FUNDP), Rue de Bruxelles 61, BE-5000 Namur, Belgium ¶[email protected] [email protected] ∗∗[email protected] Received 5 February 2010 The second-order nonlinear optical properties of two monomeric red fluorescent proteins, mStrawberry and mCherry, have been experimentally determined by frequency-resolved femtosecond hyper-Rayleigh scattering. These proteins were found to exhibit a stronger nonlinear response than the previously described eGFP, eYFP and DsRed,1 confirming the trend that fluorophores with a more extended conjugated system, or a lower-energy band gap between ground and excited state, exhibit a larger static hyperpolarizability (β0 ). Furthermore, these experimental data were complemented with quantum chemical calculations. A discrepancy was observed between experimental and theoretical results, but this could be explained by the chromophore model extracted from the available X-ray diffraction data. While eGFP showed a larger dynamic experimental response (βHRS ) due to the highest resonance enhancement, we measured an even higher signal for mCherry. Furthermore, mCherry also shows a better separation of the second harmonic signal and two-photon excited fluorescent signal, making this the preferred fluorescent protein for second harmonic imaging at 800 nm so far. Keywords: Fluorescent proteins; second-harmonic imaging; mStrawberry; mCherry; hyper-Rayleigh scattering; theory.

1. Introduction Just like any other protein, green fluorescent protein (GFP) consists of an unbranched polymer of amino acids, coupled together by peptide bonds. In all known fluorescent proteins (FPs) so far, the amino acid chain is folded as a so-called 1

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(a)

(b)

Fig. 1. (a) All known fluorescent proteins are shaped as a so-called beta barrel protecting the fluorophore in the center of the barrel. (b) The amino acids participating in the post-translational modification are marked green (Threonine 65 – Tyrosine 66 – Glycine 67), while the highlighted part represents the chromophore or fluorophore, which is the resulting extended conjugated system responsible for absorption and fluorescence in the visible spectrum.

beta-barrel (Fig. 1). The fluorescence of these proteins is due to post-translational modifications in the backbone of the amino acid chain, in the center of the barrel.2,3 The post-translational modifications give rise to a bigger conjugated system than the aromatic amino acids that already occur in the center of the protein. This special conjugated system is called the fluorophore, or more generally a chromophore, and it is responsible for a few unique optical properties, such as absorption and fluorescence in the visible spectrum. This post-translational modification seems to be independent of the organism expressing the protein, making fluorescent proteins excellent tools for biochemical research. Roger Y. Tsien and his coworkers have been very successful in creating new fluorescent proteins, mainly derivatives from the original GFP as well as from other fluorescent proteins naturally occurring in corals, e.g. DsRed,4 with new and improved properties. These fluorescent proteins have the same macromolecular structure, while the amino acid sequence can differ from three residues up to more than half of the chain. The resulting composition of the chromophore will have its effect on the spectral properties of the fluorescent protein.2–5 The general

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rule is that a longer chromophore will absorb and emit at higher wavelengths, congruently with the particle-in-the-one-dimensional-box model for the electron in a conjugated system. The energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) will be smaller in longer chromophores and since the lowest excited states are most of the times characterized by HOMO-LUMO configurations, there will be an electronic resonance at a lower frequency, hence a longer wavelength. Other important factors influencing the spectral properties can be the interactions with neighboring amino acid residues and the effect of pH on the electron distribution. Fluorescent proteins were already known to exhibit higher-order nonlinear optical properties, like two-photon absorption and two-photon excited fluorescence (TPEF). In both cases, the sum of two photons is in resonance with the chromophore, making it absorb both photons at once. This can only happen when two photons meet the molecule at exactly the same time. This can be induced by using a high-powered pulsed laser to introduce a high photon density. Another second-order nonlinear optical property, second-harmonic generation (SHG), will also occur. In this phenomenon, the photons will not be absorbed by the molecule in order to reach an excited state, but the light will be scattered. This phenomenon is also called frequency doubling, and is used in laser setups and optoelectronics. Even though the field of nonlinear optics (NLO) emerged around the same time as when the GFP was first isolated from the jellyfish Aequorea victoria,6,7 it took over 30 years for the first results on the combination of second-harmonic generation (SHG, a second-order NLO effect) and GFP to be published.8 Lewis et al. were able to image changes in membrane potential with SHG images of the worm Caenorhabditis elegans expressing membrane-targeted GFP in the neurons. Even though they proved that GFP is a useful tag for second-harmonic imaging (SHI) and that it could serve as a sensor for membrane potentials in their experiment, the intrinsic nonlinear optical properties of GFP were not characterized until recently.9 The GFP mutant eYFP was even reported not to display any second-order NLO properties.10 In 2007, Asselberghs et al. measured the first hyperpolarizability (β) of both eGFP and the photo-switching fluorescent protein Dronpa.9 Because the advantages of higher-order nonlinear optics are being more and more appreciated for biological research, this experiment was followed by measuring the first hyperpolarizabilities of eGFP, eYFP and DsRed, three commonly used fluorescent proteins in biological and biochemical research. These experiments proved that even eYFP exhibits second-order NLO properties, but confirmed that this signal is surprisingly weak, presumably due to the partial centrosymmetrical structure of the chromophore. FPs are very often used in cellular imaging because of the non-invasive and specific way to label proteins inside a cell. Nonlinear processes like TPEF and SHI add the advantage of a higher resolution since a high photon density is needed for a higher-order response. The use of longer wavelengths has the intrinsic advantage of being less damaging to biological tissue and being able to penetrate samples much

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deeper, which makes it an interesting technique for whole-animal imaging. The latter reasons make scientists more interested in using the more red-shifted FPs in single-photon imaging of biological and live samples. This incited us to also explore the nonlinear optical properties of the “mFruits”,11 an artificially obtained rainbow of monomeric derivatives of DsRed, like mOrange and mPlum, with their emission maxima between 530 nm and 650 nm. Here we report on the earliest described and most commonly used mFruits: mStrawberry and mCherry. 2. Materials and Methods 2.1. Materials The proteins were expressed and purified as described previously.9,12 After HPLC affinity purification, the samples were dialysed against a 20 mM HEPES buffer containing 150 mM NaCl at a pH of 7.5. The pRSET plasmids containing the mCherry and mStawberry gene were a generous gift from Roger Y. Tsien’s lab. The proteins were checked for purity by SDS-PAGE and mass spectrometry. Concentration of the samples was determined using the extinction coefficient of the chromophore at the wavelength of maximal absorption.11 The HRS experiments were carried out starting from 100 µM protein solutions. 2.2. Instrumental methods The determination of the linear properties as well as the hyper-Rayleigh scattering measurements were carried out using the same instruments and experimental setup as previously described.1 The HRS measurements were performed using a femtosecond pulsed Ti:sapphire laser emitting light at 800 nm. A 400 ± 10 nm filter was used to select for signal at 400 nm. 2.3. Computational methods The structures of the chromophores represented in Fig. 2 were taken from X-ray diffraction data (XRD).11 For mCherry, the positions of the H atoms were optimized at the Density Functional Theory (DFT) level of theory using the B3LYP exchange–correlation functional and the 6-31+G* basis set. Based on the values of the XRD bond lengths and bond angles, we had to consider two potential structures of the mStrawberry chromophore. The first one is the so-called mStrawberry enol form, where the enol group is stabilized by a hydrogen bond as shown in Fig. 2(b). The other model is the so-called mStrawberry alcohol form as shown in Fig. 2(c). Structure (b) was optimized like the mCherry chromophore but, since some groups display unexpected bond distances, we also optimized the group geometry under several dihedral angle constraints. Structure (c) was taken from a preliminary optimization step carried out at the PM3 level. It should be noticed that the π-conjugated system is the same for the mCherry and mStrawberry chromophores,

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(a) mCherry

(b) mStrawberry enol form

(c) mStrawberry alcohol form Fig. 2. Models for the mCherry and mStrawberry chromophores. The conjugated systems and hence the supposed chromophores are highlighted in the structures on the left side.

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which means that only geometrical constraints (differences in dihedral angles) can influence the π-electron conjugation. The dynamic first hyperpolarizability values (β) were calculated with the timedependent Hartree–Fock (TDHF)13,14 scheme while the static one was obtained by using the Coupled–Perturbed Hartree–Fock (CPHF). The TDHF calculations were carried out for wavelengths of 1064 and 1900 nm. To take into account the solvent effects, the Polarizable Continuum Model within the integral equation formalism (IEF-PCM) was used.15,16 The reported quantities are related to the actual HRS experiments with plane-polarized incident laser light and observation made perpendicular to the propagation plane. Then, the full intensity reads  2 2  + βXZZ . βHRS (−2ω; ω, ω) = βZZZ All reported β values are given in 10−30 esu. [8.641 × 10−33 esu = 1 au of β = 3.62×10−42 m4 V−1 = 3.2063×10−53 C3 m3 J−2 ] within the B convention. Although electron correlation is missing in this approach, the CPHF/TDHF scheme has often been found to provide qualitatively accurate first hyperpolarizabilities, certainly within a class of parent compounds. The vertical excitation energies [∆Ege = ωge = (ωe − ωg )] and the excited state properties of the compounds were calculated at the time-dependent DFT (TDDFT) level of approximation using the B3LYP hybrid exchange–correlation functional and the 6-311G(d,p) basis set. The solvent effects were also taken into account by using the IEF-PCM model with water as solvent. This approach has been found to provide UV-visible spectra in, at least, good qualitative agreement with respect to experimental data for both excitation energies and oscillator strengths.17–25 All calculations were performed with the Gaussian 03 package.26 3. Results The nonlinear optical properties determined by hyper Rayleigh scattering are listed in Table 1, side by side with the previously reported β values of eGFP, eYFP and DsRed.1 While βHRS in this table is the dynamic hyperpolarizability using 800 nm laser light after correction for fluorescence contribution, β0 , the static hyperpolarizability, is βHRS after an additional correction for resonance enhancement, since we are measuring at resonance conditions. When we compare the β values of the Table 1. Wavelengths of maximal absorption and fluorescence, and experimentally determined first hyperpolarizibilities of 5 fluorescent proteins currently studied. All β values in 10−30 esu.

λmax,abs λmax,flu βHRS (800 nm) β0

eGFP

eYFP

DsRed

mStrawberry

mCherry

488 nm 507 nm 107 ± 17 33 ± 5

513 nm 527 nm 49 ± 5 18 ± 2

558 nm 583 nm 81 ± 8 39 ± 4

575 nm 596 nm 104 ± 5 54 ± 4

587 nm 610 nm 134 ± 16 71 ± 14

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Fig. 3. (Color on line) Experimental (solid line) and simulated (dashed line) absorption spectra for mCherry (dark red) and mStrawberry (light red), and experimental spectra for eGFP (green line), eYFP (yellow line) and DsRed (pink line).

red FPs with the previous set of FPs, we notice that the trend in β0 confirms our expectations and, moreover, that βHRS of mCherry is larger than that of eGFP. For these specific fluorophores, we did not expect any emission at 400 nm since both the single-photon (Fig. 3) and two-photon absorption spectra27 show very low absorption at 800 nm. Our HRS measurements however seem slightly influenced by a small fluorescence contribution. We therefore executed our measurements so that we were able to perform demodulation of the apparent β to get rid of this influence.28,29 The demodulation curves are shown in Fig. 4. The experimental dynamic βHRS measurements for mCherry and mStrawberry were carried out at a wavelength that is quite far from the absorption band, so that they are only slightly impacted by resonance effects. Subsequently, the twostates approximation (TSA)30 is a good approximation to extract the static first hyperpolarizabilities from the experimental data, provided β is dominated by a single excited state. Thus, comparison between the experimental and theoretical values does not necessarily require a high-level pre-treatment of the experimental

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Fig. 4. The apparent, modulation frequency-dependent (or apparent), dynamic first hyperpolarizability βHRS , 800 for mStrawberry (•) and mCherry (). The inset shows the growing phase difference between the total HRS signal and a scattering reference.

data, contrary to our previous study of eGFP, eYFP, and DsRed.1 In this previous case, we considered 3 levels of refinement within the TSA to rule out influences by resonance effects when evaluating the static values of β from the measured dynamic responses. In the first level, the TSA assumes that only one excited state contributes to the second-order NLO response. The second level of refinement introduces a damping factor in the TSA, and the most refined level is the TSA with an inhomogeneous broadening based on the absorption spectra. This last level implicitly contains information on the distribution of the transition frequencies as well as the vibronic structure of the excited states (see Ref. 1 for more information). Nevertheless, for the sake of completeness and consistency with our previous study, we list in Table 2, the static β values as determined with the three levels of refinement of the TSA, for mCherry and for mStrawberry in comparison with the fluorescent proteins of our previous work. The parameters for mCherry and mStrawberry are listed in Table 3. As expected, the static β values of mCherry and mStrawberry are very similar for the three levels of refinement of the TSA. The relative position of the excitation energies was confirmed by the TDDFT/B3LYP calculations, which predict an excitation wavelength of 553 nm for mCherry and 546 nm for the mStrawberry alcohol form, whereas the mStrawberry enol form has an excitation wavelength of 579 nm. 4. Discussion 4.1. mStrawberry We can clearly state that the experimental results match our expectations. The FPs with a larger chromophore have a higher β0 . The computational results on

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Table 2. Comparison between calculated, measured, and extrapolated values of the first hyperpolarizabilities (10−30 esu). mStrawberry enol

mStrawberry alcohol

EGFP

EYFP

DsRed

βHRS,800 βHRS ,∞ TSA βHRS,∞ TSA (homog. damping) βHRS,∞ TSA (homog. + inhomog. damp., vibr.)

107 ± 17 33 ± 5 35 ± 6

37 ± 4 14 ± 2 14 ± 2

81 ± 8 39 ± 4 41 ± 4

104 ± 5 54 ± 3 56 ± 3

134 ± 16.5 71 ± 9 75 ± 9

19 ± 3

13 ± 2

33 ± 3

51 ± 2

70 ± 9

βHRS,∞ HF/IEFPCM (solvent = water) βHRS ,1900 HF/IEFPCM (solvent = water) βHRS,1064 HF/IEFPCM (solvent = water)

31

25

39

88

73

65

14

12

21

50

41

39

25

21

44

219

143

124

Table 3.

mCherry

Best parameters fitting the UV-vis absorption spectra.

ωge (eV); λeg (nm) γhomogeneous (eV) γinhomogeneous (cm−1 ) S ωvib (cm−1 ) γvib (cm−1 )

mStrawberry

mCherry

2.16; 573 0.174 100 0.70 1230 800

2.12; 585 0.162 100 0.50 1250 700

the other hand reveal an unexpected behavior for mStrawberry. It is important to understand that mStrawberry was not an evident sample for both the experimental measurements and the computational calculations. 4.1.1. mStrawberry in experimental measurements It is known that mStrawberry is very sensitive to pH-changes compared to many other FPs.11 Even the linear properties change dramatically upon changing the pH. Since we based our concentration measurements on the extinction coefficient of mStrawberry from literature, it is very well possible that our calculations of beta from experimental measurements are based on inaccurate concentration data. We do not expect this to be a large error since the differences in concentration √ will be less than 30%, which would only filter through to the β values as a factor 1 + 30%. In any case, the hyperpolarizability of mStrawberry will still be smaller than the result we found for mCherry. 4.1.2. mStrawberry in computational calculations Comparison between the experimental and theoretical data for mCherry and mStrawberry chromophores enables us to exclude the mStrawberry enol form.

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Indeed, going from mCherry to mStrawberry, a blue shift (+12 nm) of the wavelength of maximum absorption is expected and only the alcohol form is associated with a blue shift of the excitation energy (see Table 2). The calculated first hyperpolarizabilities for mCherry and mStrawberry do not exhibit the same order as the experimental data, which is not the case for the three other chromophores.1 As already mentioned, the π-conjugated system is highly identical for the mCherry and mStrawberry chromophores, but it differs from the other set of chromophores. Hence, the trends in the first set of 3 systems are more easily unravelled than for the two chromophores investigated in this paper. In order to understand the origin of this discrepancy between the β values, several aspects should be analyzed in more detail. The experiments were performed in aqueous solution while the calculations were carried out using XRD structures, which means on crystallized proteins. Since the π-conjugated pathway is directly influenced by the protein folding, one possible source of discrepancy can be attributed to the folding, that differs in the crystallized proteins versus aqueous solutions and is associated with different geometrical constraints. In this sense, the flawed resolution of the X-ray diffraction data represents a supplementary inconvenience when assigning the optimization constraints for the mStrawberry structures. Yet, another point that should not be ignored is the level of the calculations, which are done at CPHF-TDHF level, therefore lacking electron correlation effects. 4.2. mFruits for second harmonic imaging The aim of this research is to provide users with a list of fluorescent proteins with good characteristics for both SHG and (two-photon) fluorescence imaging. This will be very dependent on the preferred excitation wavelength and whether a strong SHG signal is required, or whether the separation between SHG and TPEF is more important. It is crucial to see the difference between β0 and the actually measured signal. While eGFP will have an effective signal that is stronger than eYFP and DsRed due to resonance enhancement and a significant contribution of fluorescence at 400 nm, the two newly measured FPs have an even stronger effective signal than eGFP without these effects. Furthermore, the TPEF signal will be able to travel further through the biological samples than light at lower wavelengths, which enables researchers to capture signal from cells deeper inside tissues. For the same reason it would be very interesting to measure first hyperpolarizabilities at wavelengths higher than 800 nm. 5. Conclusion and Perspectives Even though much more experiments are needed before we can conclude on a specific relationship between the structural, the linear and the nonlinear optical properties of fluorescent proteins, our expectations are quite well met. The simplified rule to expect a higher hyperpolarizability for a larger conjugated π-system is still an acceptable rule of thumb. Furthermore, mCherry exhibits a larger dynamic βHRS

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than eGFP for which the signal is boosted by quite some resonance enhancement. Combined with a much smaller contribution of fluorescence at 400 nm, this makes mCherry an even better suited chromophore for SHG microscopy. For future research, it is important to investigate the pH-dependency of the hyperpolarizability of the fluorescent proteins and to find methods to determine the concentrations of these proteins as accurately as possible (e.g. based on the Strickler–Berg equation relating the molecular extinction coefficient to the fluorescence radiative lifetime31 ). It is also very interesting to measure hyperpolarizabilities at wavelengths higher than 800 nm. Not only the dubious information from X-ray data, but also the observation of merely identical chromophores in mStrawberry and mCherry complicates the design of a proper model for quantum chemical calculations. Furthermore, finding the ideal balance between calculation time and inclusion of the most important surrounding residues, molecules, or perhaps even just atoms, remains a challenge for the suitable simulation of these species.

Acknowledgments INPAC is acknowledged for financial support and for the PhD fellowship for E. De Meulenaere, E. Botek thanks the IAP 6/27 for her postdoctoral grant. S. Spaepen thanks the Research Fund K. U. Leuven for his postdoctoral fellowship grant. The calculations have been performed on the interuniversity Scientific Computing Facility (iSCF) installed at the FUNDP, for which the authors gratefully acknowledge the financial support of the F.R.S.-FRFC and the “Loterie Nationale” for the convention No. 2.4617.07, and of the FUNDP. Part of this work was supported from research grants from the Belgian Government (IUAP N◦ P6-27 “Functional Supramolecular Systems”).

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