Fluorescence of Bili-Sense

Bili-Sense was excited with a 405 nm pulsed laser source (PLS-300, Picoquant, Berlin, Germany) and the fluorescence decay curves were recorded from 640 – 800 nm with a multi-channel detector (PML-16C, 16 spectral channels). In these experiments the time-resolved fluorescence of the biliverdin chromophore by direct excitation via the Soret band (S0-S2 excitation) was captured (Figure 58).

Figure 58 Fluorescence decay curves at the 710 nm emission maximum of Bili-Sense after excitation with a pulsed 405 nm laser in the absence (black curve) or presence (colored curves) of NADH (A). Decay associated spectra (DAS) of the sensor in the absence (B) or presence (C) of 1 mM NADH. The total amplitude for both components of each DAS was normalized to one (1 + 2 = 1).

Figure 58 A shows the fluorescence decay curves obtained in this experiment. The difference in amplitude for 100 µM NADH is rather small and about 20 % reduction is seen in the presence of 1 mM NADH. Figure 58 B & C exhibit the DAS for the whole spectral range from 640 to 800 nm, and indicate that biliverdin fluorescence decays nearly monoexponentially with a lifetime of 700 ps (black curve), irrespective of whether NADH is present or not. This indicates that the about 20 % reduction of the fluorescence amplitude by 1 mM NADH is due to e.g. static quenching, but without an effect on fluorescence lifetime. A small contribution of a long decay component with about 5 ns (see red curve in Figure 58 B & C) is present in both DAS shown, which only shows an increase from 5.03 to 6 ns in the presence of 1 mM NADH. The peak of the fast component in the DAS lies at around 720 nm, corresponding to the fluorescence maximum of the biliverdin

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chromophore in iRFP-like proteins. The observed differences, taken together, indicate that NADH dos not influence the lifetime of the biliverdin chromophore and the reduced amplitude may either be due to static quenching, or the effect could be explained by chromophore bleaching during the long-lasting single photon counting experiment (30 minutes per measurement).

In order to test the hypothesis, whether there is excitation energy transfer from tryptophan residues to the embedded biliverdin chromophore, and whether the emission of tryptophan residue(s) rather than that of biliverdin itself is affected by certain nicotinamides, time-resolved experiments were conducted with a 280 nm pulsed diode as excitation light source, and the fluorescence decay curves were recorded from 650 – 820 nm. The decay-associated spectra from these decay curves are shown in Figure 59.

Figure 59 DAS derived from a global fit of the fluorescence of the Bili-Sense sensor with a monoexponential model in the absence (black curve) or presence (colored curves) of NADH. Pulsed excitation was performed with a 280 nm diode laser. The DAS amplitudes were set to zero at the highest wavelength.

The DAS shown in Figure 59 verify the presence of an excitation energy transfer from tryptophan residues (directly excited by the pulsed 280 nm laser) to the biliverdin chromophore (sensitized emission recorded between 650 and 820 nm).

The black curve depicts the long wavelength emission in the absence of NADH.

Compared to 405 nm excitation, the DAS are markedly different. The maximum of

the spectrum lies at around 713 nm with two shoulders, one at 675 nm and one at 750 nm. The spectrum is of course different from the time-integrated experiments done with the Fluoromax-2 spectrometer, as the 2^nd order diffraction signal from tryptophan fluorescence is suppressed by the utilized detector (by blazed gratings), and only the true emission bands contribute to the recorded signal. Also, the lifetimes are different from those measured with 405 nm excitation, starting with 2 ns, which is markedly different from the ~700 ps fluorescence lifetime of iRFP-like proteins (seen with direct 405 nm excitation, Figure 59), and rather approximates to the characteristic lifetime of tryptophan fluorescence in aqueous solution (2.8 nm, see below) (Cao et al., 2019). If FRET occurs, the characteristics of tryptophan fluorescence (donor) would be transferred to the lifetime of the FRET acceptor (biliverdin) and determine the lifetime of the overall ET. The observation of a clear biliverdin emission band in the DAS also further clarify that the sensor signal and response is not just a spectrometer artefact, but clearly the biliverdin signal contributes to the NADH-dependent response. The band structure of the emission is conserved upon addition of NADH, while the total amplitude decreases as does the fluorescence lifetime. Concomitant with the decrease in the amplitude, the time-constant of the observed process is reduced from 2 ns in the absence of NADH to 1.53 ns in the presence of 500 M NADH, leading to a further decrease in observable fluorescence. This indicates that quenching of tryptophan fluorescence in the Bili-sense protein by NADH is due to a superposition of static and dynamic quenching (red and blue curve). The time constant observed in this experiment is theoretically constituted of the whole excitation energy transfer from the excited tryptophan to the biliverdin chromophore and subsequent emission from the chromophore. In the previously proposed model of interaction of the sensor with NADH, it was hypothesized that light excites tryptophan residue(s) in the protein backbone, which transfer their excitation energy towards the biliverdin chromophore. The chromophore accepts the energy via transfer into the Soret band, and finally emission of fluorescence from the Q band of the chromophore occurs. When NADH is present, the nicotinamide competes for this energy transfer process and offers an alternative relaxation pathway, thereby reducing the efficiency and likelihood of resonance energy transfer. Here, it is critical to determine, by which process (static or dynamic) the fluorescence of tryptophan is quenched by NADH. If the quenching process were purely static, the

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lifetime of tryptophan fluorescence would remain unchanged and only the amplitude would decrease. Thus, the fluorescence lifetime observed in the Bili-Sense experiments shown in Figure 59 hints at a superposition of these two different quenching mechanisms.