pH Dependence of Frex

The Frex sensor was constructed with a circularly permuted YFP as the chromophore (Zhao et al., 2011). Hence, the sensor inherits its spectral characteristics, like the two excitation wavelengths and a profound pH dependency of the fluorescence spectrum (Elsliger, Wachter, Hanson, Kallio, & Remington, 1999; Schwarzländer et al., 2014; Wachter, Elsliger, Kallio, Hanson, & Remington, 1998; Zhao et al., 2011). This fact was already mentioned in the introducing publication of Frex, and it was advised, in order to validate if a given signal from the sensor is in fact due to altered [NADH] and not due to altered pH, to concomitantly carry out control experiments with the isolated chromophore cpYFP. For a basic idea of the probe’s behavior at different pH levels, we chose to investigate Frex fluorescence response towards different pH levels in vitro. The F480 maxima were plotted against the applied pH value (Figure 21).

Figure 21 Fluorescence responses of the purified Frex sensor protein measured at 510 nm, expressed as the logarithm of the fluorescence amplitudes measured upon 480 nm excitation (F480), at different pH-values in the absence of NADH and NAD⁺. The fluorescence amplitudes were normalized to the maximum of F480 at pH 7. The inset shows normalized fluorescence amplitudes for the range between pH 7 and 8.5. The concentration of the sensor protein was 500 nM in PBS. Experiments were carried out at 30 °C.

Within the pH range between 7 and 10.5, the fluorescence emission of the probe enhances dramatically upon alkalization of the sample. Between pH 7 and 10.5 an enhancement factor of the fluorescence amplitude of about 45 is observed, which is about 5.5 times the dynamic range of the sensor due to NADH binding at neutral pH. These findings are supported by the literature, in which also an enormous increase of the fluorophore’s dynamic range upon alkalization of the surrounding environment was found, and the pKa of cpYFP was estimated to lie between 8.6 – 8.9, almost entirely stemming from the long wavelength excitation (Griesbeck, Baird, Campbell, Zacharias, & Tsien, 2001; Schwarzländer, Logan, Fricker, &

Sweetlove, 2011). While it is highly unlikely that the intracellular pH deviates as much as one unit from the neutral state, even small aberrations in pH have profound effects on the probe’s emission. Specifically, between pH 7 and pH 8 the fluorescence emission was enhanced by a factor of 2.5.

76 2 Frex for Intracellular Application in R. eutropha

These in vitro characterizations indicate several pitfalls regarding the use of Frex in vivo. For one, the ideally exclusive NADH sensor does in fact respond to the oxidized congener of NADH, a fact which, if not accounted for, would give ambiguous readouts, especially in the concentration range of NAD⁺ which is presumably present in the desired host R. eutropha. For another, the experimental conditions need to be inspected closely for alterations in pH, since even small intracellular pH fluctuations can rapidly generate greater changes in the fluorescence signal, than the interaction between the senor and the probe ever could.

2 Frex for Intracellular Application in R. eutropha

Frex was aimed to report on NADH levels in R. eutropha cells, expressing the soluble hydrogenase (SH). The SH couples the oxidation of hydrogen to the reduction of NAD⁺ and the generation of NADH. This elevation of NADH levels upon activity of the SH is supposed to be reported by Frex sensor. In order for the Frex protein to be expressed in the -proteobacterium, the cDNA was introduced on a plasmid vector under the control of the promotor of the soluble hydrogenase, therefore ensuring coincident expression of the SH and its activity reporter Frex.

The cultivation of R. eutropha was carried out heterotrophically, hence the cells were kept in fructose-containing growth medium for two days. Afterwards the culture was diluted to an OD435 of 0.1 in growth medium containing both fructose and glycerol in equal amounts (0.2 % each). The expression of SH and Frex is dependent on the triggering of the SH promoter. The SH promoter is activated by switching from the preferably metabolized carbon source fructose to glycerol. This switch of growth conditions from utilization of fructose to glycerol mostly occurs about one day after start of cultivation, when cells are kept at 30 °C (B. Friedrich, Heine, Finck, & Friedrich, 1981; C. G. Friedrich, 1982). The cultures are kept at this temperature, since the promoter is temperature-sensitive, repressing expression under control of this promoter at temperatures above 30 °C (C. G. Friedrich &

Friedrich, 1983).

Table 5 Characteristics of the utilized R. eutropha strains.

strain characteristics Origin

H16 Wild-type strain SH⁺, MBH⁺, RH⁺ HF500 hoxGhoxChoxH SH^–, MBH^–,

RH^–

(Kleihues et al., 2000)

HF798 hoxGhoxC SH⁺, MBH^–, RH^– (Lauterbach, 2013)

The cDNA for Frex was introduced into two different host strains, namely HF500 and HF798 (Table 5)(Kleihues et al., 2000; Lauterbach, 2013). The strain HF500 carries deletions of the genes for the large subunits of the regulatory hydrogenase, as well as the membrane-bound hydrogenase and the soluble hydrogenase and is thus not capable of expressing functional hydrogenases of these kinds. The HF798 strain is derived from the HF500 strain by re-introduction of the genes for the large subunit of the soluble hydrogenase, allowing for the functional expression of this enzyme. The HF798 strain was chosen over the wild-type strain, which expresses all of the aforementioned hydrogenases, in order to be able to connect the metabolization of hydrogen directly with a single hydrogenase, with no other conceivable side reactions. The HF500 strain itself acts as a negative control, in order to determine the effects on Frex fluorescence in response to experimental parameters not related to the SH activity. For the general experimental protocol both strains were exposed to the same stimuli and the differences in the observed behavior should enable us to infer correlations to SH activity.