Fluorescent Probes in the Local Environment of Avidin and
3.2. Aminocoumarin–Biotin Derivatives
3.2.1. Introduction
Aminocoumarins are polar push-pull fluorophores which have the great advantage that a wealth of information is available on their photophysics and solvation dynamics in the literature [55, 286, 287], on top of being photochemically stable and readily affordable. In order mainly to investi-gate their solvation dynamics in a protein environment, 6-aminocoumarin and coumarin 151 were coupled directly to biotin (AC-btn and C151-btn, respectively, Figure 3.4) and their photophysics was studied in the close environment of the proteins Avd and Stv using steady-state optical spec-troscopy and time-resolved fluorescence specspec-troscopy. Because of the absence of spacer between the chromophores and biotin, the bound dyes are located directly at the exit of the biotin binding pocket.
NH
Figure 3.4. Molecular structure of AC-btn and C151-btn.
3.2.2. Results
a) Steady-State Photophysics
Coupling of C151 and AC to biotin via an amide bond strongly affects the position of the visible absorption band by shifting it to lower wavelengths (Figure 3.5). Although the absorption spectrum of AC was not deter-mined experimentally, it should not be very different from that of 7-aminomethylcoumarin, also called coumarin 120 (C120), which has been reported to peak at 341 nm in water [289, 290].
As a consequence of this modified absorption, the fluorescence maximum of C151-btn lies at about 440 nm (Table 3.1), compared with
Table 3.1. Absorption maximum (abs, fluorescence maximum (fl, and 0–0 en-ergy transition 20-0, with 20-0"#2abs!2fl$/ 2 [288], of several coumarin deriva-tives in aqueous solution.
Dye (abs (nm) (fl (nm) 20-0 (eV)
C151 367 496 2.82
C151-btn 336 440 3.23
C120? 341 443 3.22
AC-btn 329 464 3.25
S ?Data from [289].
Figure 3.5. Intensity-normalised absorption spectra of C151, C151-btn, and AC-btn in PBS-EDTA (PE) and of C151 in acetonitrile (ACN).
495 nm for C151 in PBS-EDTA (PE). AC-btn displays a fluorescence spectrum peaking at 465 nm with a shoulder around 430 nm (Figure 3.6).
The fact that the designed coumarin-biotin constructs cannot be effi-ciently excited at 400 nm prevents using them in fluorescence up-conversion studies with the set-up available in the laboratory (see section 6.2.3). Polar solvation dynamics at the protein interface was thus studied with another probe (Lucifer Yellow, see section 3.4). Steady-state and nanosecond fluorescence dynamics investigations were nonetheless per-formed with AC-btn and C151-btn in the presence of Avd or Stv.
The presence of the protein does not significantly alter the position of the absorption band of the constructs (Figure 3.7). A slight hypochrom-ism can be detected with C151-btn and a weak hyperchromhypochrom-ism with AC-btn. Changing the number of protein binding sites occupied on average between 1 and 4 does not either induce significant changes in the spec-trum, within experimental errors. Furthermore, the spectra are very simi-lar for each probe in Avd and in Stv. The additional absorption appearing below 320 nm stems from the protein (Figure 3.9).
The fluorescence spectrum of C151-btn is hypsochromically shifted upon binding to Avd and Stv with respect to the free dye (Figure 3.8). It gets broader and a shoulder appears around 450 nm. With AC-btn, the Figure 3.6. Intensity-normalised fluorescence spectra of C151, C151-btn, and AC-btn in PBS-EDTA (PE) and of C151 in acetonitrile.
Figure 3.7. Absorption spectra of various equivalents of C151-btn (A, B) and AC-btn (C, D) bound to Avd (A, C) or Stv (B, D) in aqueous buffer solution. The spectrum of the free dye is shown for comparison (dashed line). The legend of (B), (C), and (D) is the same as in (A).
Figure 3.8. Intensity-normalised fluorescence spectra of various equivalents of C151-btn (A, B) and AC-btn (C, D) bound to Avd (A, C) or Stv (B, D) in aque-ous buffer solution. The spectrum of the free dye is shown for comparison (dashed line). The legend of (B), (C), and (D) is the same as in (A).
original shoulder at about 430 nm becomes much more pronounced when the construct is bound to either protein. On the other hand, the red wing of the spectrum gets narrower. Both effects are more pronounced in Figure 3.9. Intensity-normalised absorption spectra of Avd, Stv, and biotin in PBS-EDTA. As an indication of the relative intensities, ,282 = 96’000 cm!1&M!1 with Avd, ,282 = 224’000 cm!1&M!1 with Stv, ,264 = 280 cm!1&M!1 with biotin.
Table 3.2. Fluorescence quantum yield >fl of C151, C151-btn, and AC-btn in ACN, PBS-EDTA, and in the host proteins at various dye/protein ratios (dye equivalents per protein tetramer, eq.).
Environment >fl(C151) >fl(C151-btn) >fl(AC-btn)
ACN 0.57? 0.12 0.02
PBS-EDTA 0.35? 0.20 0.04
Avd, 1 dye eq. 0.005 0.010
Avd, 2 dye eq. 0.004 0.008
Avd, 3 dye eq. 0.004 0.007
Avd, 4 dye eq. 0.004 0.009
Stv, 1 dye eq. 0.003 0.006
Stv, 2 dye eq. 0.003 0.005
Stv, 3 dye eq. 0.003 0.005
Stv, 4 dye eq. 0.003 0.005
? Data from [287].
Stv than in Avd. Again, the number of occupied sites has no influence on the spectra.
The fluorescence quantum yields were determined against C151 in ACN (0.57 [287]) (Table 3.2). The quantum yield of C151 is lower in aqueous buffer solution than in ACN. Coupling to biotin further reduces the fluorescence quantum yield to 0.20. This quantity is even 5 times lower with AC-btn. Upon binding to Avd or Stv, the fluorescence gets strongly quenched, with quantum yield values between 0.003 and 0.010.
b) Nanosecond Fluorescence Dynamics
The excited-state lifetime of the free and bound dyes was measured using the TCSPC technique upon excitation at 395 nm (Figure 3.10, Figure 3.11). Since the dye/protein ratio, which was varied between 1 and 4, does not significantly influence the decays, only traces for a dye/protein ratio of 2 are given and considered to be representative.
C151 displays a single-exponential fluorescence decay both in ACN and in aqueous buffer solution (Table 3.3). When the dye is coupled to Table 3.3. Time constants 6( used to reproduce the fluorescence decays of C151, C151-btn, and AC-btn in various environments, as well as average lifetimes 6av and radiative lifetimes 6rad. Relative amplitudes different from 1 are given below the lifetime value in brackets.
Figure 3.10. Nanosecond fluorescence dynamics of C151, C151-btn, and AC-btn in PBS-EDTA and of C151 in acetonitrile measured with a 420 nm cutoff filter.
Figure 3.11. Nanosecond fluorescence dynamics of C151-btn, and AC-btn in PBS-EDTA and in the presence of Avd or Stv (2 dye equivalents per protein tetramer).
biotin, the fluorescence decay becomes biphasic with one dominant component (98 % in amplitude) around 1.6 ns. Upon binding of C151-btn to Avd and Stv, the dynamics become highly non-exponential. The dominant component (> 95 % in Avd, > 98 % in Stv) has a lifetime shorter than 200 ps and can thus not be resolved with the TCSPC appara-tus. The radiative lifetime is highly enhanced, which is indicative that a quenching process actively takes place with these systems.
Coupling of AC to biotin has a dramatic effect on the excited-state dy-namics of the dye (the excited-state of which is expected to decay mono-exponentially): three exponential terms are required to accurately repro-duce the measured excited-state decay of AC-btn. The dominant compo-nent (> 98 %) has a lifetime which cannot be resolved in the experiment, indicating that some highly efficient intramolecular fluorescence quench-ing process is operative. Although the workquench-ing concentration was low, aggregation of the dyes due to poor dye solubility in water cannot be to-tally ruled out. When AC-btn is bound to Avd or Stv, the average lifetime increases, although the fluorescence quantum yield decreases, suggesting the occurrence of a new quenching process.
3.2.3. Discussion
a) Effect of Biotin on the Photophysics
Linking AC and C151 via their amine moiety to biotin strongly affects the photophysics of the two chromophores. It induces a hypsochromic shift of the absorption maximum in aqueous solution, of more than 2500 cm!1 in the case of C151. Aminocoumarins are push-pull chromophores and their first electronic transition has strong charge transfer character from the electron rich amino to the electron withdrawing carbonyl group in polar solvents [287, 291]. They usually exhibit absorption maxima around 370 nm and above, depending on the electron-donating ability of the amino group which can be increased by introducing @-alkyl substituents and by rigidifying it through cyclisation as in the well-studied coumarin 153. Unsubstituted coumarins or coumarins bearing electron-withdrawing substituents such as alkoxyl groups absorb at lower wavelengths, generally around 340 nm and below [290]. On the other hand, C(O)—N(H) amide bonds are known to have partial double-bond character because of
delo-calisation of the nitrogen lone pair over the C—N bond (this usually causes the four atoms of the amide moiety to be coplanar). This reduces the availability of the nitrogen lone pair for the charge transfer transition, causing a strong hypsochromic shift of the absorption band. AC-btn and C151-btn then essentially behave as electron-poor coumarin dyes.
The coupling to biotin also has only a minor effect on the excited-state photophysics of C151. The fluorescence spectrum of C151-btn is blue-shifted with respect to that of C151, its fluorescence partially quenched, but the deactivation of the excited state takes place almost monoexponen-tially with a dominant lifetime which is also reduced (the radiative rate constant is thus similar to that of C151 in ACN, a solvent which has a refractive index very similar to water). With AC however, the effect of biotin on the photophysics is much more pronounced. Although antici-pated to peak at a lower wavelength, the fluorescence spectrum of AC-btn appears to be red-shifted with respect to that of C120 [289] which has its maximum at 443 nm; the fluorescence quantum yield markedly de-creases, and the excited state decays with high non-exponentiality. These features point toward an intramolecular quenching process which is likely to occur, at least partially, because the nature of the electronic transition is affected in the biotin constructs. Although no explanation to it seems to have been proposed so far, this effect has been known for several decades [292, 293] and used to develop protease assays based on fluorogenic aminocoumarin peptide substrates which are specifically cleaved by a protease, thus releasing the fluorescent aminocoumarin [293-295].
Alternatively, self-quenching by formation of aggregates due to low solubility of the construct in water can also be considered. The fact that the fluorescence spectrum is shifted to higher wavelengths with respect to C120 and displays a shoulder around 430 nm where it is expected to be would support this hypothesis. Indeed, one could imagine that two spe-cies contribute to the fluorescence spectrum: one which is not aggregated and has its fluorescence maximum around 430 nm, another that is aggre-gated and the fluorescence of which is shifted to higher wavelengths, as observed with other chromophores forming H-type dimers (see sections 1.4.3 and 4.4). A distribution of geometries is certainly expected and could explain the non-exponentiality of the fluorescence dynamics. To avoid the strong perturbation exerted by biotin on the photophysics of the
two aminocoumarins, a good try would be to achieve the coupling of a coumarin derivative to biotin through an exocyclic position which is not involved in the lowest electronic transition.
b) Effect of the Protein on the Photophysics
The presence of both Avd and Stv influences the photophysics of C151-btn and AC-C151-btn. The fluorescence quantum yield of the biotin derivatives and the radiative rate constant strongly decrease upon binding to either protein. The quenching is a little more pronounced in Stv than in Avd.
Photoinduced electron transfer between the coumarin moiety and a Trp residue of the protein should account for this since the proteins have several Trp residues in the binding pocket region as shown in Figure 3.1.
Indeed, the excited-state energy for both coumarin derivatives is around 3.25 eV and the sum of the oxidation potential of Trp (1.03 V vs NHE [296]) and the reduction potential of C120 (–1.90 V vs NHE in DMF [289]), which should not be very different from that of AC-btn or C151-btn in water, gives 2.93 eV. With an additional stabilisation due to the aqueous environment of X 0.1 eV [289], a driving force for the photoin-duced electron transfer of roughly –0.4 eV can be estimated.
Figure 3.12. Fluorescence quenching of C151 by Trp in water.
The possibility of quenching C151 by Trp is confirmed by a bimol-ecular quenching experiment in water in which Trp was progressively added to a C151 solution. The resulting Stern-Volmer plot (Figure 3.12) shows that the fluorescence intensity of C151 diminishes upon increasing Trp concentration, much more than the fluorescence lifetime. This is indicative that a large part of the quenching occurs in a static way, mean-ing that a ground-state complex is formed between C151 and Trp. The planar '-system could indeed favour a stacking interaction between the molecules. A dynamic quenching rate constant of 7.4 10 sZ 9 -1&M!1 is extracted from the slope of the Stern-Volmer plot. As the rate constant of diffusion in water is of the order of 6.5 10 sZ 9 -1&M!1 [297], one can con-clude that the quenching is highly efficient. Although the driving force is rather low, the photoinduced ET reaction between the C151 chromo-phore and Trp in the protein is expected to be ultrafast since the two partners do not need to diffuse on a long distance before the reaction can occur because they are preorganised through formation of the biotin-host protein complex. Trp has been shown to be able to quench several other organic dyes [54, 298, 299].
3.2.4. Conclusions
Two aminocoumarins have been coupled to biotin with the hope that these polarity probes would be suited to study the effect of a protein inter-face on their excited-state dynamics. However, the constructs do not ab-sorb at 400 nm, a prerequisite for investigating the early fluorescence dynamics of the dyes with the used laser system, because the absorption and emission bands are shifted to lower wavelengths with respect to the uncoupled dyes. Steady-state and nanosecond-resolved investigations of the photophysics of C151-btn and AC-btn have shown that the properties of the chromophores are strongly altered when biotin is coupled to the amino substituent of the coumarins and that the modified dyes essentially behave as electron-poor coumarins. The reason for this alteration is not clear and a full understanding would go beyond the scope of this study, but the fact that the nature of the electronic transition changes due to involvement of the amine nitrogen lone pair in a partial electron delocali-sation over the C—N amide bond when the dyes are coupled to biotin certainly plays an important role. Linking coumarins to biotin through
other additional substituents on the coumarin rings without interfering with the lowest energy electronic transition could help overcoming this problem.
Furthermore, the fluorescence of these biotin derivatives is strongly quenched when they are bound to both Avd and Stv, suggesting that photoinduced electron transfer takes place between the dyes and a Trp residue. This possibility is experimentally supported by the observed quenching of a C151 solution by Trp.
Overall, C151-btn and AC-btn can certainly report on the presence of Avd and Stv in solution due to the strong quenching of the fluorescence, but the fact that their excited-state properties are strongly altered upon binding to their host proteins and that the ultrafast fluorescence quench-ing process is likely to take place, at least partially, on the time scale of solvation dynamics, the process which was initially intended to be fol-lowed, precludes the use of these constructs to achieve the original goals of this study. The main reason for this is that the aminocoumarins are too readily reduced.