Fluorescence Spectroscopy

For further characterization of the Bili-sense protein, the sensor’s fluorescence excitation and emission spectra were recorded (Figure 55). For the recording of the spectra the samples were concentrated to a total protein concentration of 4

M, corresponding to 220 nM holoprotein.

144 5.4 Bili-Sense

Figure 55 Excitation spectrum of the designed sensor Bili-sense (A). The emission wavelength was set to 750 nm and the spectrum was recorded over the shown wavelength range. Emission spectrum of Bili-Sense after excitation at 320 nm in the short (340 – 630 nm) and long (660- 900 nm) wavelength region (B).

Comparison of the emission spectra after excitation with 320 nm in the short (red curve) and long (black curve, corresponding to 2^nd order diffraction signal of tryptophan fluorescence) wavelength region of the spectrum show an about 12 times higher fluorescence intensity in the short wavelength region. Shown in the inset is the emission spectrum in the long wavelength region on an enlarged scale.

Concentration of Bili-Sense was adjusted to 4 M (OD280) in PBS buffer.

In a first set of experiments, excitation spectra with emission detection set to 750 nm were recorded, since the biliverdin chromophore is known (from properties of iRFP713) to emit in this region (Figure 55 A). These spectra indicated an excitation maximum of the protein around 320 nm, the wavelength that was previously used as excitation of iRFP713 for the NADH titrations. When 320 nm light was used for excitation, the spectrum shown in Figure 55 B was recorded, which exhibits a very broad emission band centered around 750 nm.

This band seems to be only composed of the 2^nd order diffraction of the tryptophan fluorescence in the used Fluoromax-2 instrument, which was already observed in the iRFP713 spectra. In contrast to the iRFP713 fluorescence spectra, no distinct chromophore peak at 713 nm was observed, and recording of the emission spectra in the wavelength region between 300 and 500 nm (Figure 55 B) showed clear characteristics of tryptophan fluorescence, which verified that the spectral features in the red region are merely due to the 2^nd order diffraction of tryptophan

fluorescence. The intensity of the spectra in the first order diffraction (around 380 nm) and second order diffraction (around 750 nm) varies about 12-fold.

In order to determine whether the sensor carried the biliverdin chromophore, the protein was also investigated by excitation at 660 and 680 nm, which should excite an embedded biliverdin chromophore via the Q band (Figure 56).

Figure 56 Emission spectra after excitation with 660 nm (black curve) and 680 nm (red curve). Concentration of Bili-Sense was adjusted to 4 M (OD280) in PBS buffer.

The emission spectra shown in Figure 56 exhibit the specific biliverdin emission peak for both excitation wavelengths positioned at 710 nm, very close to the emission peak of the parental iRFP713 (Filonov et al., 2011). This indicates that the purified sensor is at least partially chromophorylated in order to detect biliverdin-specific fluorescent traces.

In order to improve chromophorylation of the protein and thereby enhancing the contribution of the biliverdin fluorescence within the broad background of 2^nd order diffraction around 750 nm of Bili-Sense holoprotein, the expression was carried out at lower temperatures as well (compare Table 7). The corresponding proteins were purified from these cultures and they were analyzed by fluorescence spectroscopy (Figure 57).

146 5.4 Bili-Sense

Figure 57 Emission spectra in the wavelength region between 690 – 900 nm after excitation at 320 nm for Bili-Sense purified from cultures kept either at 37 °C (black curve), 30 °C (red curve) or 21 °C (blue curve) (A). Emission spectra in the wavelength range between 690 – 800 nm after excitation at 680 nm for the three different expression temperatures, 37 °C (black curve), 30 °C (red curve) and 21 °C (blue curve) (B). Protein concentration was adjusted to 4 M (OD280) in PBS.

When the protein purified from cultures grown at 21 °C or 30 °C was investigated, apparent differences appeared in the respective spectra, most noticeably upon excitation with 320 nm and 660 nm (Figure 57). In Figure 57 A the emission spectra upon excitation at 320 nm for proteins obtained from three different expression temperatures are shown. The spectrum for the protein expressed at 37 °C shows the already discussed broad 2^nd order diffraction of tryptophan fluorescence centered around 750 nm. The broad fluorescence emission is also observable for protein purified from expression cultures grown at 30 °C; however, the intensity of the fluorescence is slightly weaker. The protein purified from an expression culture grown at 21 °C exhibits the lowest contribution of the 2^nd order diffraction signal. Notably, both spectra of the protein expressed at 21 °C and 30 °C show a pronounced shoulder or even a small peak at 713 nm, indicative of the biliverdin chromophore. This indicates that the emission in the red spectral range can at least partially be attributed to the biliverdin chromophore and not just to the spectrometer artefact.

In Figure 57 B the emission spectra of the corresponding proteins are plotted after excitation at 680 nm. All three spectra show the characteristic 710 nm band resulting from the direct excitation of the chromophore. However, notably, the

spectrum of the protein from 30 °C expression exhibits an intensity about seven times higher than the protein sample retrieved from cultures kept at 37 °C. The protein retrieved from the 21 °C expression shows a medium intensity upon excitation at 680 nm. These findings suggest that the protein sample retrieved from cultures kept at 37 °C is only marginally consisting of holoprotein, with the majority being made up of apoprotein. The 2^nd order diffraction signal of tryptophan fluorescence dominates the spectrum after excitation at 320 nm, while in the spectra of the other two expression conditions a discernible shoulder at 713 nm is observable. The spectra obtained upon 680 nm excitation support these findings and show that the protein obtained from the culture grown at 30 °C incorporated the highest amount of chromophore and exhibited the largest holoprotein fraction.

However, even with the slightly optimized chromophorylation in the samples from the 30 °C cultures, the signal in the red wavelength region upon 320 nm excitation will mainly be comprised of the 2^nd order diffraction of tryptophan fluorescence.

In order to test if the newly designed sensor showed a specific answer towards NADH that was detectable as a change in fluorescence lifetime, the protein was examined in a different spectrometer for time-resolved fluorescence detection.

This setup is equipped with a blazed grating that suppresses the 2^nd order diffraction of tryptophan fluorescence emission. The time-resolved spectra obtained with this spectrometer showed a biliverdin-specific signal at 713 nm upon 320 nm excitation (vide infra), which thus confirmed that some part of the signal around 750 nm in the spectrometer without blazed grating must contain signatures of a biliverdin-specific signal.