Fluorescent Probes in the Local Environment of Avidin and
3.4. Lucifer Yellow–Biotin Derivatives
3.4.1. Introduction
A biotin derivative labelled with LY, which is commercially available (Lucifer Yellow biocytin, LY-13-btn), was bought and used as such (Figure 3.26). In order to see the influence of the position of the probe with respect to the protein on the ultrafast fluorescence dynamics, LYen was also directly coupled to biotin via an amide bond (LY-3-btn). The influence of Avd and Stv on the photophysics of these two dyes was stud-ied by steady-state and time-resolved absorption and fluorescence tech-niques. Except the fluorescence up-conversion experiments which were performed with 2 dye equivalents (eq.), all measurements were done with
1–4 dye equivalents. For the sake of simplicity however, only traces with 2 equivalents are shown, since the dye/protein ratio did not significantly affect the photophysics.
3.4.2. Results
a) Influence of Biotin on the LY Chromophore Photophysics
In order to be sure that biotin does not significantly affect the photophys-ics of the LY chromophore as it did with coumarin dyes, a few simple absorption and emission properties of LY-3-btn and LY-13-btn were compared with those of LYen in various solvents. Neither the position, nor the shape of the absorption and fluorescence spectra were signifi-cantly modified in the biotin constructs (Figure 3.27). The fluorescence quantum yield and lifetime slightly increased in aqueous solution and essentially remained unaffected in DMF and DMSO (Table 3.8), suggest-ing that the efficiency of the excited-state proton transfer mechanism ob-served with LYen (see section 3.3) is somewhat reduced with the biotin constructs. Overall, these straightforward experiments ensured that LY-3-btn and LY-13-LY-3-btn had the characteristics required for the planned
Figure 3.26. Molecular structure of LY-3-btn and LY-13-btn.
Figure 3.27. Absorption and fluorescence spectra of LY-3-btn, LY-13-btn, and LYen in PBS-EDTA.
Table 3.8. Photophysical properties of LYen, LY-3-btn, and LY-13-btn in differ-ent solvdiffer-ents: position of absorption, (abs, and emission, (em, maxima, fluores-cence quantum yield >fl, excited-state lifetime 6fl, and radiative lifetime 6rad.
Solvent Dye (abs (nm) (em (nm) >fl 6fl (ns) 6rad (ns)
H2O LYen 428 536 0.20 5.7 27.8
LY-3-btn 427 539 0.24 7.3 30.7
LY-13-btn 426 542 0.24
PBS-EDTA LYen 427 538 0.20 5.6 27.6
LY-3-btn 427 537 0.23 6.9 30.6
LY-13-btn 426 541 0.24 6.9 28.5
D2O LYen 428 539 0.40 11.5 28.6
LY-3-btn 428 539 0.41 12.7 30.6
DMSO LYen 439 520 0.46 10.6 23.0
LY-3-btn 434 519 0.47 11.8 25.2
DMF LYen 435 514 0.47 11.3 24.1
LY-3-btn 433 514 0.47 12.1 25.9
b) Steady-State Photophysics of the Bound LY-Biotin Constructs The absorption spectra of LYen, LY-3-btn, and LY-13-btn are nearly identical and centred at about 427 nm in aqueous solution (Figure 3.27, Table 3.8). Binding to Avd or Stv only affects its position and intensity to a minor extent (Figure 3.28): 10–20 % hypochromism can be observed, except for LY-3-btn in Stv; the spectrum does not move with LY-3-btn
Figure 3.28. Absorption spectra of LY-3-btn (left) and LY-13-btn (right) in PBS-EDTA and when bound to Avd (dotted line) or Stv (dashed line).
Figure 3.29. Fluorescence spectra of LY-3-btn (left) and LY-13-btn (right) in PBS-EDTA and when bound to Avd (dotted line) or Stv (dashed line).
and Avd, whereas it shifts by 4 nm to lower wavelengths with LY-3-btn and Stv, and by 3–4 nm with LY-13-btn in Avd and Stv.
The intensity-normalised fluorescence spectra of the free dyes in aqueous solution are almost identical. The position of the fluorescence spectrum of LY-13-btn is not affected by the presence of either protein (Figure 3.29), whereas it is hypsochromically shifted by 10 nm with LY-3-btn in Avd, which is consistent with a less polar environment. Indeed, a blue-shift is also observed with free LY-3-btn and LYen in DMSO and DMF (see Table 3.8).
The fluorescence quantum yield (Table 3.9) of free 3-btn and LY-13-btn is identical both in water and in PBS-EDTA (0.23) and 1.2 times higher than that of LYen in water (0.20). Upon binding to Avd, the fluo-rescence of LY-3-btn slightly increases (0.26), which might reflect a less polar or a less proton-rich environment (again, the same behaviour is observed with the free dye and LYen in DMSO or DMF (see Table 3.8), whereas it decreases in the case of bound LY-13-btn (0.14). Binding to Stv leads to a reduction of the fluorescence quantum yield of both LY-3-btn (0.17) and LY-13-LY-3-btn (0.09). Overall, it seems that Stv quenches to some extent the fluorescence of both LY-13-btn constructs and Avd only slightly that of LY-13-btn.
Table 3.9. Fluorescence quantum yield >fl, excited-state decay times 6( (and amplitudes), average lifetime 6av, and radiative lifetime 6rad of 3-btn and LY-13-btn free in PBS-EDTA and when bound to Avd or Stv.
System >fl 67 (ns) 68 (ns) 6av (ns) 6rad (ns)
LY-3-btn 0.23 6.9 (1.00) 6.9 30.6
LY-3-btn Avd 0.25 3.5 (0.17) 8.3 (0.83) 7.5 29.9
LY-3-btn Stv 0.17 2.5 (0.19) 6.5 (0.81) 5.7 33.0
LY-13-btn 0.24 6.9 (1.00) 6.9 28.5
LY-13-btn Avd 0.14 1.2 (0.20) 7.1 (0.80) 5.9 43.8 LY-13-btn Stv 0.09 1.4 (0.08) 6.5 (0.92) 6.1 65.9
LYen 0.20 5.6 (1.00) 5.6 27.6
c) Nanosecond Fluorescence Dynamics
The excited-state lifetime was measured using the TCSPC technique (Figure 3.30) and found to be identical (6.9 ns) for both free probes.
Upon binding to either protein, the fluorescence decay can be well re-produced using a biexponential function (Table 3.9). The biexponential decays might in fact reflect a distribution of lifetimes due to a distribution of conformations of the probe-protein complex. The presence of a faster decay component with bound LY-13-btn is however suggested from the initial bend in the kinetics. This hypothesis is strengthened by the fact that the radiative rate constant calculated from the fluorescence quantum yields and the average fluorescence lifetimes is identical for all systems except for bound LY-13-btn and maybe LY-3-btn in Stv, indicating that a faster decay component has probably been missed. Photoinduced elec-tron transfer might account for this ultrafast component and for the de-crease of the fluorescence quantum yield.
To ensure that the binding of the LY-biotin constructs specifically oc-curs through biotin in the biotin-binding pockets, LYen was added to an aqueous solution containing Avd. Neither the spectral features, nor the fluorescence quantum yield and lifetimes were significantly affected with respect to LYen in aqueous buffer solution.
Figure 3.30. Fluorescence decay of LY-3-btn (left) and LY-13-btn (right) in PBS-EDTA or when bound to Avd or Stv. The legend is the same in both parts of the figure.
d) Ultrafast Fluorescence Dynamics
The early fluorescence dynamics of both probes was investigated using the fluorescence up-conversion technique. For each system, the decays were monitored at 8–9 wavelengths over a broad range of the fluores-cence spectrum (Figure 3.31 and Figure 3.32) and were analysed globally by a sum of one Gaussian and three exponential functions (Table 3.10).
The amplitudes were then adjusted to match those of the steady-state fluorescence spectrum (Figure 3.33 and Figure 3.34) and time-resolved emission spectra (Figure 3.35) were reconstructed by fitting a log-normal function to the experimental data points. The centre of this log-normal function was used to express the absolute Stokes shift of the dye, 1abs
# $
$ (see equation (1.58)), as a function of time (Figure 3.36). Since the solu-tions contained 5 % (v/v) DMSO as a co-solvent (the dye stock solusolu-tions were made in DMSO), the early fluorescence dynamics of LYen in such PBS-EDTA-DMSO mixture was reinvestigated in this environment for comparison.16With all systems, a Gaussian component (61) of about 200 fs had to be included to accurately reproduce the initial rise of the fluorescence.
Without this component, an unrealistically large IRF would have had to be
16 No difference in the steady-state properties or in the nanosecond fluorescence dynamics was seen between LYen in PBS-EDTA or in the PBS-EDTA-DMSO mixture.
Table 3.10. Lifetimes obtained from the fitting procedure on the early fluores-cence dynamics of btn, LY-13-btn, and LYen in PBS-EDTA and of LY-3-btn and LY-13-LY-3-btn when bound to Avd and Stv (dye/protein ratio of 2). 61 corre-sponds to the Gaussian component.
Figure 3.31. Early fluorescence dynamics of LY-3-btn in PBS-EDTA or when bound to Avd and Stv at 480, 495, 510, 530, 550, 570, 590, 610, and 630 nm from bottom to top for each data set (dye/protein ratio of 2).
Figure 3.32. Early fluorescence dynamics of LY-13-btn at 490, 510, 530, 550, 570, 590, 610, and 630 nm in PBS-EDTA and Avd and at 480, 495, 510, 530, 550, 570, 590, and 610 nm in Stv, from bottom to top for each data set (dye/protein ratio of 2).
assumed. The amplitudes associated with 6 62- 4 are positive at short wave-lengths and negative at long wavewave-lengths. This corresponds to a red shift of the fluorescence band with time. Additionally, components with posi-tive amplitude at all wavelengths (6 65- 6), therefore related to an acceler-ated decay (fluorescence quenching) of the excited-state population, were found with both constructs in Stv and with LY-13-btn Avd. The same systems also have a reduced fluorescence quantum yield compared with the free dyes (see above). Photoinduced electron transfer between the LY chromophore and a nearby Trp residue of the protein might account for this observation.
To visualise the temporal evolution of the spectral shape of the fluo-rescence more easily, time-resolved emission spectra (TRES) were recon-Figure 3.33. Wavelength dependence of the amplitude factors obtained from the global analysis of the fluorescence dynamics of LY-3-btn in PBS-EDTA and bound to Avd or Stv (dye/protein ratio of 2).
structed from the time profiles recorded at the different wavelengths.
Such intensity-normalised spectra are displayed in Figure 3.35 for LY-3-btn in PBS-EDTA and in Stv. Although the number of wavelengths is relatively small, it clearly appears that the spectra shift to higher wave-lengths with time, which is consistent with a time-resolved Stokes shift.
The centre of the reconstructed TRES was used to follow the evolu-tion of the fluorescence band with time (Figure 3.36). Two to three expo-nential functions are required to describe the dynamics of this parameter and the found time constants are similar to those used to describe the fluorescence dynamics (Table 3.11). The fractional amplitudes of the different components are plotted for each system in Figure 3.37. It is Figure 3.34. Wavelength dependence of the amplitude factors obtained from the global analysis of the fluorescence dynamics of LY-13-btn in PBS-EDTA and bound to Avd or Stv (dye/protein ratio of 2).
Figure 3.35. Reconstructed intensity-normalised time-resolved emission spectra of LY-3-btn in PBS-EDTA and in Stv between 0.3 and 100 ps after excitation.
The solid lines represent best log-normal fits to the experimental data points.
Time delays are: 0.3, 0.5, 1, 2, 3, 5, 10, 20, 30, 50, and 100 ps after excitation.
The steady-state spectra are shown as well (dashed line, hidden by other traces in PBS-EDTA as the red shift is much faster than in Stv).
Figure 3.36. Time evolution of the peak position of the fluorescence spectrum of the investigated systems. The inset is a zoom on the first 10 ps.
clear from this plot that if the presence of the proteins does not seem to strongly affect the time constants associated with the solvent relaxation processes, the relative amplitudes of the slower processes rise, meaning that the dynamics is slowed down in a protein environment.
The decay of the fluorescence polarisation anisotropy of the free dyes takes place monoexponentially on a time scale of a few hundreds of pico-seconds, as expected for molecules of this size in water (Table 3.12, p.
140). When LY-3-btn and LY-13-btn are bound to Avd or Stv, only a partial depolarisation was observed on a 40 ns time window (Figure 3.38),
PBS-EDTA Avd Stv PBS-EDTA Avd Stv
0.00 0.25 0.50
0.75 6s1
6s2
6s3
LY-13-btn LY-3-btn
Fractional Stokes shift amplitude
Figure 3.37. Fractional amplitude of the Stokes shift components observed in the fluorescence dynamics of the investigated systems.
Table 3.11. Lifetimes, 6s(, and relative amplitudes (in brackets) extracted from the temporal evolution of the Stokes shift observed with the different systems depicted in Figure 3.36.
System 6s1 (ps) 6s2 (ps) 6s3 (ps) LY-3-btn 1.4 (0.69) 5.1 (0.23) 123 (0.07) LY-3-btn Avd 1.0 (0.45) 6.7 (0.42) 82 (0.13) LY-3-btn Stv 2.2 (0.71) 21 (0.29)
LY-13-btn 1.5 (0.64) 5.3 (0.27) 73 (0.09) LY-13-btn Avd 0.6 (0.35) 3.0 (0.39) 32 (0.26) LY-13-btn Stv 1.4 (0.62) 9.9 (0.38)
LYen 0.9 (0.60) 4.1 (0.40)
indicating that the dyes indeed bind to their host protein. Depolarisation due to rotational diffusion of the protein is expected to take place on a time scale of tens of nanoseconds. Nonetheless, a fraction of the anisot-ropy was found to decay on a time scale similar to that of the free dyes, pointing out that the chromophores still have some intrinsic rotational freedom around the spacer which links them to biotin when the con-structs are bound to the protein. This biphasic behaviour was analysed in terms of the “wobbling-in-a-cone” model (see below).
e) Transient Absorption
In order to try to establish whether photoinduced electron transfer be-tween the LY chromophore and the protein might cause the reduction of the fluorescence quantum yield and the fast decay components moni-tored in the fluorescence dynamics of the LY-biotin constructs in Stv, Figure 3.38. Fluorescence anisotropy decay measured with the different systems at 550 nm (circles). The solid lines represent best fits using the “wobbling-in-a-cone” model (see section 3.4.3).
transient absorption experiments with LY-13-btn were performed (Jakob Grilj). Transient spectra of LY-13-btn in aqueous buffer solution obtained upon 400 nm excitation (Figure 3.39) display three distinct features which evolve with the same time constants over a nanosecond time scale: a tive band between 420 and 480 nm due to ground-state bleaching, a nega-tive band between 480 and 630 nm due to stimulated emission, and an excited-state absorption above 630 nm.
When LY-13-btn (200 +M) is mixed in aqueous solution with Trp at 40 mM concentration, the same three bands are qualitatively observed but important differences are nonetheless noticed (Figure 3.39). Apart from an artificial signal appearing in the course of the measurement in the very blue edge of the spectra and hiding the ground-state bleach, the
ex-Figure 3.39. Transient absorption spectra of LY-13-btn in PBS-EDTA (top), in the presence of 40 mM Trp (middle), and bound to Stv (bottom) recorded upon excitation at 400 nm.
cited-state decay is highly accelerated as suggested by the decrease of the excited-state absorption and stimulated emission bands. This indicates that LY-13-btn is quenched by Trp, as expected from investigations of LYen in the presence of Trp (see section 3.3). Furthermore, the band ascribed to stimulated emission is narrower on its red side and the ex-cited-state absorption band broader on its blue side than when Trp is absent, suggesting that an additional absorption band overlaps in the 580 nm region. This absorption can be safely ascribed to the Trp radical cation, Trp(0 [355] (see section 3.3). A time-profile of the absorption band at 580 nm (Figure 3.40) indeed reveals that after an ultrafast in-crease due to formation of Trp(0 with a subpicosecond time constant, the 580 nm signal decays with a 3.3 ps time constant, very close to the 2.6 ps measured with LYen under very similar conditions (see Figure 3.25). The plateau value reached in the transient absorption signal decays on the nanosecond time scale and arises from residual stimulated emission of the order of 1 ps due to solvation dynamics, the transient time profile of
Figure 3.40. Time profile of the transient absorption signal at 580 nm extracted from the spectra shown in Figure 3.39. The solid lines are best single exponential fits to the data points.
the absorption at 580 nm of LY-13-btn in the absence of Trp consists only of this nanosecond component.
Besides the ground-state bleach and the excited-state absorption also monitored with the dye free in solution, transient absorption spectra re-corded with LY-13-btn bound to Stv (Figure 3.39) also display a some-what narrower stimulated emission band. Time profiles of the absorption at 580 nm exhibit however only a rise with a 26 ps time constant and a nanosecond component. The 26 ps time constant corresponds to an ac-celerated decay of the excited-state and can thus be related to the 8.1 and 100 ps decays observed in the fluorescence dynamics.
3.4.3. Discussion
a) Solvation Dynamics
Components in the fluorescence dynamics with associated time constants 62–64 can clearly be associated with a red shift of the fluorescence spec-trum with time, as demonstrated by their decay-associated amplitudes which change sign from positive to negative with increasing wavelength.
They are responsible for the observed dynamic Stokes shift (Figure 3.35 and Figure 3.36). With the free LY-3-btn and LY-13-btn, three time scales are involved in the Stokes shift: X 1–2 ps, X 4–5 ps, and X 70–
120 ps. The 1 ps component can be safely ascribed to the solvation dy-namics induced by the diffusive motion of bulk water (see section 1.3.2).
Time constants close to 1 ps have already been observed with many other probes, among which LYen, in water [50, 53, 54].
The 4–5 ps component represents roughly 25 % of the Stokes shift of LY-3-btn and LY-13-btn. It is also present with LYen in the water-DMSO mixture but not with LYen in pure water, which indicates that it is related to DMSO. The reported time constants for the diffusive solvation dynam-ics of coumarin 153 in DMSO are 2.3 and 10.7 ps [55] with an average value weighted by the relative amplitude of these two components of 3.8 ps, which agrees rather well with the observations made here. The amplitude of about 25 % of this component with the LY-biotin derivatives and even 40 % with LYen does however not correspond to the only 5 % volume fraction, that is, 1.3 % mole fraction of DMSO used. From this, it can be concluded either that the probes are preferentially solvated by
DMSO molecules or that this component has another — unknown — origin related to the presence of DMSO in the solution. Molecular dy-namics simulations with explicit solvent molecules support the first hy-pothesis by showing that the solvation dynamics of coumarin 153 is dominated by DMSO even in low DMSO content DMSO-water mixtures because of the preferential solvation of the probe [356]. Additional ex-periments with the same systems in pure aqueous solution would further help validating or rejecting this conclusion.
A third component representing less than 10 % of the total amplitude was necessary to describe the evolution of the Stokes shift of free LY-3-btn and LY-13-LY-3-btn. Because the fluorescence dynamics was recorded only up to 160 ps after excitation, the value of this component should be taken rather with caution, although the time scale of several tens of pico-seconds can be taken as accurate. It was not observed with LYen. Com-ponents of the solvation dynamics longer than 10 ps have been reported for probes at biomolecular interfaces (see Chapter 2), but never for sim-ple chromophores in aqueous solution. At interfaces with a protein, such time scale has been ascribed to solvation by the protein itself which un-dergoes motions coupled to reorganisation of the water network [160, 199]. In the case of LY-3-btn and LY-13-btn, it is not unrealistic to imag-ine that, due to the relatively large size of the molecules, some internal structural dynamics might take place after optical excitation of the LY chromophore, especially if the biotin and LY parts interact by dipolar or hydrogen-bonding couplings. Calculation of the structure of the mole-cules in an aqueous environment could give some credit to this hypothe-sis. The peptide melittin, consisting of 26 amino acids, displays a slow solvation component of 87 ps [177] and apparently even very short pep-tides of only a few amino acid residues also exhibit solvation dynamics on this time scale (D. Zhong, personal communication). Other possibilities can however not be ruled out.
When the LY-biotin constructs are bound to Avd or Stv, the solvation dynamics slows down, as expected with probes at protein interfaces (see section 2.3). With LY-3-btn in Avd, three time scales very similar to those seen with the free ligand are observed. The amplitude of the component attributed to bulk water however strongly decreases and that of the two slowest components rises by a factor X 2. This can be interpreted as the
probe being located right at the protein surface at the exit of the biotin
probe being located right at the protein surface at the exit of the biotin