Rigid Rod-Based FRET Probes for Membrane Sensing Applications

Article pubs.acs.org/JPCB

Rigid Rod-Based FRET Probes for Membrane Sensing Applications Ursula Eisold,† Nicole Behrends,‡ Pablo Wessig,‡ and Michael U. Kumke*,† †

Physical Chemistry and ‡Bioorganic Chemistry, University of Potsdam, Institute of Chemistry, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany ABSTRACT: Oligospirothioketal (OSTK) rods are presented as an adjustable scaffold for optical membrane probes. The OSTK rods are readily incorporated into lipid bilayers due to their hydrophobic backbones. Because of their high length-over-diameter aspect ratio, only a minimal disturbance of the lipid bilayer is caused. OSTK rods show outstanding rigidity and allow defined labeling with fluorescent dyes, yielding full control of the orientation between the dye and OSTK skeleton. This allows the construction of novel Förster resonance energy transfer probes with highly defined relative orientations of the transition dipole moments of the donor and acceptor dyes and makes the class of OSTK probes a powerful, flexible toolbox for optical biosensing applications. Data on steady-state and time-resolved fluorescence experiments investigating the incorporation of coumarin- and [1,3]dioxolo[4,5-f ][1,3]benzo-dioxole-labeled OSTKs in large unilamellar vesicles are presented as a show case.

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INTRODUCTION The cell membrane is probably the most important biological boundary, shielding the interior of a cell from the outside medium and being the first contact for external stimuli. Subsequently, the cell membrane (and its different components like proteins or sugar moieties) is the primary partner for a manifold of different interactions with the outside medium. Different signaling pathways, such as chemical, electrical, and mechanical, can be operative in triggering biochemical processes inside a cell. To monitor the interaction on a molecular/cellular level, specific tailored probes are required. Fluorescence spectroscopy is one of the leading experimental techniques in the investigation of cellular processes. The success of fluorescence-based techniques for the analysis of cellular processes is unmatched by other techniques due to the outstanding sensitivity of the methods and the plethora of tailored fluorescence probes that allow the investigation of biochemical processes with extreme spatial as well as time resolution down to the single-molecule level.1−3 Among the different classes of fluorescence probes, the concept of nonradiative resonant energy transfer based on dipole−dipole interaction between two fluorophores, which is commonly known as Förster resonance energy transfer (FRET), is frequently applied to monitor the distance between two molecules (donor (D) and acceptor (A)). The FRET concept is often referred to as a molecular ruler, allowing the analysis of conformational changes in (bio)macromolecules or indication of a binding event, for example, between an antigen and antibody.4,5 For sensing of biochemical processes, intrinsic as well as extrinsic fluorophores are applied as donor and acceptor pairs for FRET. In addition to the overall specificity of the probes used (e.g., specific localization in the system under © XXXX American Chemical Society

investigation), the fundamental performance of a FRET pair is defined by its specific spectroscopic parameters, summarized by its critical Förster distance, R0. Here, R0 determines the distance working range of the FRET pair and is used as a reference parameter to compare different FRET pairs. It describes the distance between the D and A at which the efficiency of the FRET is 50%. As a rule of thumb, distance alterations in the range of 0.5R0 < R < 2R0 can be monitored by a specific FRET pair. The combination of different intrinsic (spectroscopic) properties of the D and A determines the value of R0 (see eq 11). The overlap integral, J, is defined by the spectral overlap between the D emission and A absorption spectra and is a frequently used parameter to improve R0 (see eq 12). Also, the fluorescence quantum yield of D has been addressed to push R0 forward. The influence of the orientation factor, κ2, has been recognized, and its influence on the observed FRET efficiency has been extensively discussed.6−8 However, attempts to tailor this parameter are sparse. For a D−A couple in solution, both free to rotate, κ2 will be 2/3. In cases with D and/or A linked to a (macro)molecule, the rotational freedom can be distinctly altered.9−12 Depending on the nature of the linker and the chemical microenvironment created by the (macro)molecule, limitations in the accessible space due to intramolecular interactions or conformational hindrance can lead to distinct alterations in the value of κ2. The influence of κ2 on the calculated D−A distance can be large, and miscalculation of κ2 can lead to up to a 40% error in the calculation of R0.6,8 The situation may become even more complex in cases in which the Received: July 20, 2016 Revised: August 24, 2016

A

DOI: 10.1021/acs.jpcb.6b07285 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Chart 1. Overview of the Single- and Double-Labeled OSTK Rods Including Also the Parent Compounds

sensing on the basis of fluorescence are presented. Single- and double-labeled OSTK rods containing a coumarin (Cou) or/ and [1,3]dioxolo[4,5-f ][1,3]benzo-dioxole (DBD) dye were investigated first in phosphate-buffered saline (PBS) with respect to their fundamental photophysical parameters. From comparison with the respective parent compounds, the influence of the OSTK scaffold on the photophysics of the dye molecule was evaluated. In the second step, the OSTK probes were incorporated into large unilamellar vesicles (LUVs), which were used as simple biomimetic systems for cell membranes. Changes in the photophysics of the probes upon incorporation into LUVs were monitored. Especially, double-labeled OSTK rods carrying Cou and DBD dyes as well as a flexible linked Cou−DBD FRET pair were characterized in PBS and LUVs with respect to their FRET performances in the two different molecular environments.

κ2 value and D−A distance are no longer independent.13 Therefore, a tailored alignment (orientation as well as distance) of the D and A in a FRET pair is of great interest. Molecular rods,14−19 that is, molecules with a large aspect ratio, are increasingly used in various applications, for example, in biosciences and material sciences. In this context the rigidity, solubility, and synthetic accessibility of the molecular rods are important parameters influencing their applicabilities. Recently, we developed a new class of molecular rods based on spirocyclically joined six-membered rings. The rings are connected by condensation of 1,3-diols and ketones and therefore we called these rods oligospiroketals (OSK).20 OSK rods exhibit a high degree of rigidity compared with other molecular rods,21 and flexible combinations of different building blocks allow the synthesis of rods with different lengths. However, longer OSK rods suffer from decreasing solubility. This problem could be circumvented by incorporating solubility-enhancing groups in the middle of the rods (called “sleeves”).21,22 These OSK rods have been successfully used as anchors in biological membranes,23−25 linkers in FRET pairs,26 and building blocks in novel porous materials27 and dendrimers.28 Another limitation of OSK rods is the maximum number of ketal moieties. The reversible formation of these functional groups significantly complicates the synthesis of longer rods. The replacement of oxygen by sulfur, giving the considerably more stable thioketals, solves this problem and is the basis of the recently introduced oligospirothioketal (OSTK) rods.29 The FRET pairs used in this study were previously characterized in different organic solvents with respect to their fundamental photophysical properties.30 To use the OSTK rods for sensing applications in life sciences, aqueous systems were investigated. The recent results showing the performance of the OSTK rod as a scaffold for membrane

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EXPERIMENTAL SECTION

In Chart 1, the different single- and double-labeled OSTK rods investigated in the present work are depicted. Their synthesis has been described in detail elsewhere.22,29,30 In the different rod probes, Cou and DBD were used as dyes. Whereas Cou was attached only in the rigid binding motif, the DBD dye was attached via short but flexible linkers (“f” forms, see Chart 1). Parent compounds and single-labeled rods were used as reference systems to monitor the influence induced by changes in the molecular environment of the probes free from the contribution of FRET. Vesicle Preparation. Phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3phospho-L-serine (DOPS) were purchased from Avanti Polar Lipids, Inc. for the preparation of LUVs. Stock solutions of 10 B

DOI: 10.1021/acs.jpcb.6b07285 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B τ⎞ 1 1⎛ = ⎜1 + ⎟ ⎝ Φ⎠ r r0

mg/mL were prepared in chloroform. The solvents were obtained from Sigma-Aldrich and were of analytical grade. Stock solutions of the OSTK probes were prepared in methanol and acetone, with a concentration of 10−3 M. For the preparation of LUV samples, first a lipid film of DOPC and DOPS in a ratio of 4:1 was prepared. For this purpose, 100 μL of the DOPS stock solution was pipetted into 400 μL of the DOPC stock solution and diluted with an additional 500 μL of chloroform. After the addition of 1 μL of the OSTK stock solution, the mixture was mixed by shaking and dried in a stream of nitrogen. For thorough removal of chloroform, the lipid film was evaporated for 1 h under high vacuum. The lipid film was hydrated in 2 mL of PBS so that the resulting total lipid concentration was 2.5 g/L and 3.15 mol/L. The mixture was heated in a drying oven at 40 °C for 1 h. After at least five freeze and thaw cycles, the solution was pressed at least 35 times through a membrane of a miniextruder (Avanti Polar Lipids), with a 100 nm diameter. The resulting OSTK concentration was 5 × 10−7 mol/L. Absorption and Steady-State Fluorescence Spectroscopy. The absorption spectra were measured from 250 to 400 nm for the Cou-labeled probes, 350 to 550 nm for the DBDlabeled probes, and 250 to 550 nm for the double-labeled OSTK rods using a Lambda 750 UV/vis spectrometer (PerkinElmer). Steady-state fluorescence measurements were performed using a Fluoromax-4 spectrofluorometer (HORIBA Jobin Yvon) operated in the SPC mode. The Cou-labeled probes were excited at λex = 340 nm, and the DBD-labeled probes were excited at λex = 450 nm. The samples with the double-labeled rods were excited at λex = 340 nm and λex = 450 nm. The emission spectra were measured in the spectral range of 350 nm < λem < 500 nm for the Cou-labeled probes, 460 nm < λem < 700 nm for the DBD-labeled probes, and 350 nm < λem < 700 nm for the double-labeled OSTK rods. The fluorescence spectra were measured at a 90° angle to the excitation light and were corrected with a reference signal for any fluctuation of the excitation light intensity as well as by a quantum correction for the wavelength-dependent sensitivity of the detector channel. For the determination of the fluorescence anisotropy, the samples were excited with vertical polarized light and the fluorescence intensity (I) was detected with the polarizer set to horizontal (Ivh) and vertical (Ivv) orientations. The polarized emission was recorded at λem = 420 nm as well as at λem = 600 nm. G=

Ihv Ihh

Time-Resolved Fluorescence Spectroscopy. Fluorescence decays were measured using an FL920 spectrofluorometer (Edinburgh Instruments), equipped with a multichannel plate (Europhoton) and operated in the timecorrelated single-photon-counting (TCSPC) mode. A picosecond pulsed diode laser, EPL-375 (Edinburgh Instruments), with λex = 375 nm, a pulse width of 55 ps, and a repetition rate of 10 MHz, was used to excite the Cou-labeled probes. A supercontinuum source, SC400-PP (Fianium), with a pulse width of 30 ps and a 5 MHz repetition rate was used for excitation of the DBD-labeled probes at λex = 450 nm. The fluorescence decays were recorded with an emission polarizer (Glan Thompson prism) in the magic angle position (55°) at λem = 420 nm for the Cou-labeled probes and λem = 600 nm for the DBD-labeled probes. The data on time-resolved fluorescence intensities (I(t)) were analyzed by monoexponential or biexponential decay kinetics according to eq 4 using the commercial software package provided by Edinburgh Instruments (FAST software) for the determination of the fluorescence decay time(s), τi. n

I (t ) =

⎛ t⎞ ⎟ + const ⎝ τi ⎠

∑ αi exp⎜− i=1

(4)

For time-resolved anisotropy, the G factor was determined according to eq 5 by measuring the corresponding decays for 1200 s. G=

Ihv(t ) Ihh(t )

(5)

The time-resolved anisotropy, r(t), was calculated according to eq 6. The samples were excited with vertically polarized light at λex = 375 nm for the Cou-labeled probes and λex = 450 nm for the DBD-labeled probes. The emission was detected horizontally (Ivh(t)) and vertically (Ivv(t)) collecting photons in the TCSPC mode for 1200 s, respectively. Emission was recorded at λem = 420 nm for the Cou-labeled probes and λem = 600 nm for the DBD-labeled probes. r (t ) =

I vv(t ) − GI vh(t ) I vv(t ) + 2GI vh(t )

(6)

If a molecule is restricted in its rotation, for example, by incorporation into a phospholipid membrane, incomplete depolarization may occur and a limiting anisotropy, r∞, is observed. Moreover, the molecule may rotate only in cone angle θC (0° ≤ θC ≤ 90°), and in that case, the time-resolved anisotropy may be described by the wobble-in-a-cone model (eq 7).33−35 Using eq 7, fundamental anisotropy r0, limiting anisotropy r∞, and correlation time Φ can be calculated.

(1)

From eq 1, the G factor was determined, which corrects the anisotropy spectrum for the polarization dependence of the detection channel. Emission was measured with the excitation polarizer oriented horizontally and the emission polarizer oriented vertically (Ihv) and horizontally (Ihh) relative to the excitation polarization. Anisotropy r was calculated according to eq 2.31 I − GI vh r = vv I vv + 2GI vh

(3)

⎛ τ⎞ r(t ) = (r0 − r∞) exp⎜ − ⎟ + r∞ ⎝ Φ⎠

(2)

(7)

Hindrance of the rotation can be described by A∞ and is a function of cone angle θC (eq 8).

The correlation of the steady-state anisotropy with rotational correlation time Φ, fluorescence decay time τ, and fundamental anisotropy r0 of the fluorophore is given by the Perrin equation (eq 3).32

A∞ = C

r∞ ⎤2 ⎡1 = ⎢ cos θC(1 + cos θC)⎥ ⎦ ⎣2 r0

(8)

DOI: 10.1021/acs.jpcb.6b07285 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Table 1. Spectroscopic Properties of D Parent Compounds Cou and Cou−OSTK λexc [nm] Cou PBS Cou LUV Cou−OSTK PBS Cou−OSTK LUV

344 344 346 346

± ± ± ±

2 2 2 2

λem [nm] 416 414 416 408

± ± ± ±

2 2 2 2

τ [ns] 4.1 3.8 4.1 3.4

± ± ± ±

φ

0.1 0.1 0.1 0.1

0.58 0.64 0.34 0.71

For a strong hindrance of rotation, A∞ approaches 1 and θC is near 0°. In the case of cone angle θC being 90°, A∞ is near 0 and the fluorophore is free to rotate.31 FRET. FRET efficiencies, Eexp, were calculated with eq 9 using the fluorescence quantum efficiencies of D in absence (φD) and in the presence of A (φDA) as well as the fluorescence decay times of the D in absence (τD) and in the presence of the A (τDA). φ τ Eexp = 1 − DA = 1 − DA φD τD (9)

± ± ± ±

0.03 0.03 0.02 0.04

r

r∞

0 ± 0.02 0 ± 0.02 0 ± 0.02 0.08 ± 0.02

0 ± 0.02 0 ± 0.02 0 ± 0.02 0.1 ± 0.02

Φ [ns]

θC [°]

± ± ± ±

50 ± 5

0.3 0.2 0.3 0.2

0.1 0.1 0.1 0.1

influence of solvent polarity on the spectral positions of the absorption and emission maxima is similar.30,36 With Cou as the parent compound, the absorption maximum in buffer was observed at 344 ± 2 nm and the corresponding fluorescence maximum, at 412 ± 2 nm, which is in good agreement with those in other 6,7-dialkoxycoumarins.37−39 Addition of the OSTK unit did not significantly alter the absorption and fluorescence spectra. For the Cou−OSTK compound, no distinct shift was found in the absorption (or excitation) or fluorescence spectrum (see Table 1 and Figure 1).

The theoretical FRET efficiency, Etheor, was calculated according to eq 10, with interdye distance R and Förster distance R06,31 Etheor =

R 06 R 06 + R6

(10)

The Förster distance was calculated using eq 11; depends on the spectral overlap of the fluorescence emission of the D and absorption of the A; and is quantitatively expressed by the overlap integral (J(λ)) as well as the quantum yield of the D (φD), the relative orientation of the transition dipoles of the fluorophores (κ2), and the refractive index of the medium (n).6,31 ⎛ 9(ln 10)κ 2φ J(λ) ⎞1/6 D ⎟⎟ R 0 = ⎜⎜ 5 4 128 π N n ⎝ ⎠ A

(11)

Figure 1. Absorption and emission (λex = 340 nm) spectra for Cou (black) and the Cou−OSTK rod (blue) in PBS.

The overlap integral can be calculated according to eq 12, with the normalized intensity of the fluorescence emission of the D (FD(λ)) and the extinction coefficients of the A (εA(λ)). J (λ ) =

■

∫0

∞

FD(λ)εA (λ)λ 4 dλ

From the time-resolved fluorescence data, no alteration in the decay kinetics was found either. Moreover, comparison of the steady-state anisotropy values in buffer for Cou and Cou− OSTK shows that there is also no difference in the molecular rotation of the two molecules, as the overall size of the OSTK rod is still small, allowing a full depolarization within the fluorescence decay (see Table 1). This is complemented by the time-resolved depolarization measurements, which show no difference in the rotation correlation time (Φ = 0.3 ns) as well as in the limiting anisotropy values (r∞ = 0) of both compounds. In the presence of LUVs, the spectral characteristics of Cou− OSTK remained nearly unchanged. In the fluorescence emission spectra (λex = 340 nm), only a slight hypsochromic shift for the Cou−OSTK rod is observed upon switching from PBS (λmax = 416 nm) to LUVs (λmax = 408 nm). However, the fluorescence decay time, τ, was decreased more distinctly. For 6,7-alkoxycoumarins, the formation of an intramolecular charge transfer state upon electronic excitation was identified as the reason behind the hypsochromic shift.37,40 In Cou−OSTK, a push−pull system between the electron-rich ligand at position 7 (D) and the lactone carbonyl group (A) is operative. The Cou unit in the electronically excited state, with its increased dipole moment, will be better stabilized in polar solvents (which has

(12)

RESULTS AND DISCUSSION First, the fluorescence of the parent compounds (Cou and Cou−OSTK for the D part as well as DBD, DBD−f-OSTK, and DBD−OSTK for the A part) was measured in PBS buffer and after incorporation into LUVs (see Chart 1 for compounds). The effect of the OSTK rod on the fluorescence properties of the D as well as the A dye was characterized. In the case of the DBD dye (A), a flexible as well as a rigid attachment of the OSTK rod was investigated to elucidate the possible differences due to the binding motif. In addition to the fluorescence quantum yield (φ) and fluorescence decay time (τ) measurements, the fluorescence depolarization was recorded and used for the determination of the fluorescence anisotropy (r) and rotational correlation time (Φ) to further characterize the incorporation into the LUVs. Spectroscopic Characterization of the Parent Compounds Cou and Cou−OSTK. Cou and Cou−OSTK may be seen as members of the 6,7-dialkoxycoumarin family. Compared to that in other 6,7-dialkoxycoumarins the observed D

DOI: 10.1021/acs.jpcb.6b07285 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 2. Fluorescence excitation (λem = 420 nm) and emission (λex = 340 nm) spectra for the Cou−OSTK rod in PBS (black) and LUVs (red) (left) and their respective fluorescence decay curves (λex = 340 nm, λem = 420 nm) (right).

Figure 3. Fluorescence excitation (λem = 600 nm) and emission (λex = 450 nm) spectra for DBD, DBD-f-OSTK, and DBD−OSTK in PBS (left) and their respective fluorescence decay curves (λex = 450 nm, λem = 550 nm) (right).

been shown by the solvent dependence of the fluorescence quantum yield as well as the fluorescence decay time for 6,7alkoxycoumarins).41 The result of the increased stabilization in polar solvents is a change in the energy of the 1(ππ*) fluorescent state relative to that of the near first triplet state 3 (nπ*).42 In nonpolar (less polar) solvents, the 1(ππ*) state lies just above the 3(nπ*) state, so the intersystem crossing (ISC) rate is high. In polar solvents, the energy of the respective 1 (ππ*) state is lower than that of the 3(nπ*) triplet state, making the ISC less effective.43 The fluorescence decay curves (λex = 340 nm, λem = 420 nm) of the Cou−OSTK rods (see Figure 2) show a monoexponential decay kinetics in PBS as well as incorporated in LUVs. The fluorescence decay time of the Cou−OSTK rod in LUVs (τ = 3.4 ns) is shorter than that in PBS (τ = 4.1 ns). Considering the amphiphilic character of the Cou−OSTK molecule, it is tempting to assume that the OSTK unit is incorporated into the lipid double layer, whereas the Cou part is located in the interfacial region of the LUVs. This leads to an overall situation, in which the Cou−OSTK molecules are located in a less polar environment and, as a consequence, an altered stabilization of the different electronic states induces a decrease in the fluorescence decay time. The successful incorporation of Cou−OSTK into LUVs is further supported by the fluorescence depolarization measurements. From the steady-state anisotropy as well as from the

rotational correlation time (see Table 1), it can be seen that both parameters are increased compared to those in PBS. The limitation in the rotational freedom of the Cou−OSTK molecule is due to the incorporation into the LUV double layer. For the analysis of time-resolved anisotropy, the wobblein-a-cone model (eq 7) was applied. Here, it was assumed that the fluorophore is restricted in its rotation due to the incorporation into the lipid layer and can only rotate in a cone with a defined cone angle (eq 8). The rotational correlation time (Φ = 0.2 ns) for the Cou−OSTK rod is very fast and almost equal to that for the Cou−OSTK rod in PBS. But the rotation is hindered, and the anisotropy decay curve reaches a limiting anisotropy value (r∞ = 0.1), yielding a cone angle of about θC = 50°. This underlines that the Cou− OSTK rod is successfully incorporated into the hydrophobic bilayer of the LUVs. Photophysical Characterization of Single-Labeled DBD−OSTK Rods. The DBD dye was attached to the OSTK rod unit in a flexible (DBD-f-OSTK) or rigid (DBD− OSTK) binding motif (see Chart 1). In Figure 3, the fluorescence excitation (λem = 600 nm) and emission (λex = 450 nm) spectra of DBD, DBD-f-OSTK, and DBD−OSTK in PBS are shown. The high sensitivity of the spectroscopic properties of DBD dyes toward solvent polarity has been reported before.44 Compared to those of organic solvents like chloroform, the absorption spectra and especially E

DOI: 10.1021/acs.jpcb.6b07285 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Table 2. Spectroscopic Properties of the DBD−OSTK Rod λexc [nm]

λem [nm]

DBD PBS DBD LUV DBD-f-OSTK PBS

448 ± 2 448 ± 2 437 ± 2

612 ± 2 612 ± 2 565 ± 2

DBD-f-OSTK LUV DBD−OSTK PBS DBD−OSTK LUV

440 ± 2 441 ± 2 434 ± 2

570 ± 2 555 ± 2 558 ± 2

τ [ns] 6.4 7.0 1.4 4.9 8.2 7.3 11.1

± ± ± ± ± ± ±

0.1 0.1 0.1 (77%) 0.1 (23%) 0.1 0.1 0.1

r∞

Φ [ns]

θC [°]

0.01 ± 0.001 0.01 ± 0.001 0.04 ± 0.002

φ

0 ± 0.02 0 ± 0.02 0 ± 0.02

0 ± 0.02 0.05 ± 0.02 0 ± 0.02

0.1 ± 0.05 0.2 ± 0.1 0.1 ± 0.05

60 ± 5

0.17 ± 0.009 0.04 ± 0.002 0.32 ± 0.016

0.06 ± 0.02 0 ± 0.02 0.09 ± 0.02

0.07 ± 0.02 0 ± 0.02 0 ± 0.02

0.8 ± 0.1 0.3 ± 0.1 0.2 ± 0.1

the fluorescence spectra for DBD show significant bathochromic shifts. The Stokes shift (Δλ) for DBD (Δλ = 124 nm; λabs = 427 nm, λem = 551 nm) in chloroform is already very large, and in PBS, it is increased even further to Δλ = 164 nm (λabs = 448 nm, λem = 612 nm). In principle, a large Stokes shift was observed for DBD-f-OSTK as well as for DBD−OSTK. However, unlike that in the case of Cou, the binding of the OSTK unit to DBD has a more distinct influence on some of its photophysical properties. Upon coupling with the OSTK rod, the Stokes shift is decreased. For DBD-f-OSTK and DBD− OSTK, Δλ = 128 nm and Δλ = 114 nm were obtained, respectively, which are smaller compared to that for the parent compound. Whereas for the absorption maxima only a slight hypschromic shift was found (437 nm for DBD-f-OSTK and 441 nm for DBD−OSTK, respectively), a much stronger hyspochromic shift was observed in the emission spectra of both compounds (565 and 555 nm for DBD-f-OSTK and DBD−OSTK, respectively, compared to 612 nm for DBD), which consequently was the main reason for the distinctly decreased Stokes shift (see Table 2). The fluorescence decay of DBD followed monoexponential kinetics, with a fluorescence decay time of τ = 6.4 ns in PBS (see Figure 3). The binding motif of the OSTK rod also affected the fluorescence decay kinetics. Whereas the fluorescence decay of DBD-f-OSTK in PBS shows biexponential kinetics, with two fluorescence decay times τ1 = 1.4 ns (77%) and τ2 = 4.9 ns (23%), for DBD− OSTK, monoexponential kinetics, with τ = 7.3 ns, which was slightly increased compared to that for DBD, was found. For DBD as well as the DBD-labeled probes, no fluorescence anisotropy was found, which is caused by the fast rotational motion of the probes in a homogeneous solution. Compared to that in PBS, incorporation into LUVs had only little effect on the locations of the absorption and emission maxima. However, the fluorescence decay times and quantum efficiencies were changed. Here, the largest influence was found for DBD−OSTK, for which an increase in τ from 7.3 to 11.1 ns and an increase in φ from 0.04 to 0.32 was determined on incorporation into LUVs. Successful incorporation was also indicated by the distinct increase in the anisotropy parameters (see Table 2). Here, for DBD−OSTK, no cone angle θC could be determined from the anisotropy data. Compared with DBDf-OSTK, an additional side chain was present at the DBD part of the molecules (see Chart 1), which may alter the overall incorporation of the molecule into the LUVs. Here, our work in progress using modified OSTK rods may shed light on this unexpected observation. Investigation of FRET in Double-Labeled OSTK Rods. From Figure 4, it can be seen that the fluorescence emission of Cou−OSTK (D) shows a good spectral overlap with the absorption of DBD−OSTK (A).

r

55 ± 5

Figure 4. Absorption and emission spectra for the Cou−OSTK rod (λex = 340 nm (blue)) and the DBD−OSTK rod (λex = 450 nm (red)) in PBS.

On the basis of the D emission (Cou−OSTK) and A absorption, the spectral overlap integral, J, was calculated. The calculations were performed for Cou−f-DBD, Cou−OSTK−fDBD, and Cou−OSTK−DBD to consider the changes in the spectral data induced by the polarity of the solvent as well as attachment to the rods. Parameter κ2 is related to the relative orientation of the transition dipole moment of Cou and DBD in space and is defined by the dot product of the respective unit vector or by the respective angles (see eq 13). Depending on the connection of the DBD unit to the rod (rigid vs flexible), an orientation factor of κ2 = 3.9 (near the theoretical maximum of 4) for Cou−OSTK-DBD, where the orientation of the dipol moments is collinear, or κ2 = 2/3 for Cou−OSTK−f-DBD as well as for Cou−f-DBD (dynamic averaging possible because of the flexible connection between the D and A) was used in the calculations. The results are summarized in Table 3. κ 2 = (cos θT − 3 cos θD cos θA)2

(13)

The theoretical Förster distance for Cou−OSTK-DBD was calculated by eq 11, with κ2 = 3.9, and determined to be R0 = 4.1 nm; the R0 values for Cou−OSTK−f-DBD and Cou−fDBD were distinctly smaller. Here, the reduced κ2 value was the determining factor, as the overlap integrals of both compounds were similar or slightly larger. Compared to those of frequently used FRET pairs, the R0 values were at a lower end and in the range of those for commonly used dye pairs such as fluorescein and rhodamine (4.5 ≤ R0 ≤ 5.6 nm).14 On the basis of the structures, the distances between the centers of the two dyes for the different rods were calculated (assuming an extended configuration, which neglects any possible movement for the flexible rod probes Cou−f-DBD and Cou−OSTK−f-DBD). It F

DOI: 10.1021/acs.jpcb.6b07285 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Table 3. Summary of the FRET Parameters of the Different Double-Labeled OSTK Rods in PBS spectral overlap integral J [cm6]

Förster distance R0 [nm]

(theoretical) distance between D and A [nm]

theoretical FRET efficiency

4.5 × 10−15 5.0 × 10−15 4.2 × 10−15

3.3 3.2 4.1

1.5 2.2 1.9

0.99 0.9 0.99

Cou−f-DBD κ2 = 2/3 Cou−OSTK−f-DBD κ2 = 2/3 Cou−OSTK−DBD κ2 = 3.9

Table 4. Fluorescence Decay Times and Quantum Efficiencies as Well as the Experimental FRET Efficiencies and Distances for the Different Double-Labeled Probes E (theo) Cou−f-DBD PBS

0.99

Cou−f-DBD vesicle Cou−OSTK−f-DBD PBS

0.9

Cou−OSTK−f-DBD vesicle Cou−OSTK−DBD PBS Cou−OSTK−DBD vesicle

0.99

τ (Cou) [ns]