The opsin domain of Cop5 enables light color-signaling
Our previous study showed that Cop5 can be expressed in oocytes of X. laevis but that no guanylyl cyclase activity can be detected whereas Cop6 (Cr2c-Cyclop1) was demonstrated to be a light-inhibited guanylyl cyclase [8]. Sequence alignment shows that the GC domain of Cop5 is missing several key residues required for metal ion binding, base recognition, ribose-orienting, and transition state-stabilization in the cGMP production process (Additional file 1: Figure S1). Therefore, three chimeras of Cop5 and Cr2c-Cyclop1 were designed for fusion of corresponding fragments at regions of high homology and outside predicted domains. All three chimeras retained the complete opsin part of Cop5.
The chimeras were fused:
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1)
After the opsin domain of Cop5 (Chimera 1, C1),
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2)
Before the histidine kinase domain of Cr2c-Cyclop1 (Chimera 2, C2),
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3)
After the histidine kinase domain of Cop5 (Chimera 3, C3) (Fig. 1).
The chimeras were expressed in X. laevis oocytes and guanylyl cyclase (GC) activities of membrane extracts were measured in an established in-vitro assay [8, 12, 16]. Chimeras C1 and C2 showed a low GC activity in the dark and in 473 nm light but higher GC activity with 380 nm illumination (Fig. 2a and b). The ratio of activity under these preliminary test conditions of UV-A and blue illumination was determined to be 15 and 12 for C1 and C2, respectively.
Chimera C3 showed only a low GC activity, compared to C1 and C2, with no difference between the dark, UV-A, and blue light conditions (Fig. 2c). This implies that the His kinase domain from Cop5 is not functional or not compatible with the response regulator domain of Cr2c-Cyclop1. For further investigation, we focus on C1 because of its light-sensitivity with the highest activation ratio.
The engineered chimera C1 is bistably regulated by UV-A and blue/green light
After confirming the UV-activated GC activity of C1, we designed experiments to test whether C1 showed a bi-stable character, as might be predicted from the spectroscopic studies [17,18,19,20]. As shown in Fig. 3a, a short (30 s) 380-nm light pulse could activate the GC activity of C1 which continued in the dark. We then tested the inhibition effect with light of different wavelengths on UV-A-activated switch-Cyclop1. A 30-s (380 nm) UV-A illumination was applied in the beginning, and then the samples were either kept in the dark or in 473 nm blue light, 532 nm green light, 593 nm orange light, or 635 nm red light to measure GC activity. As shown before, the GC activity remained high in the dark. The UV-A-evoked GC activity can be efficiently turned down by 4.8 μW/mm2 473 nm blue and 532 nm green light (Fig. 3a). A 593 nm light can only turn down the evoked GC activity partially while the 635 nm red light has no effect on UV-A-evoked GC activity, similar to the dark (Fig. 3a). After a short UV-A light pulse for 30 s, the evoked GC activity could then be switched off by 30 s 473 nm light similar with the GC activity in the dark, while the activation by UV-A can be repeated after the inhibition by blue light (Fig. 3b). Figure 3b shows that C1 is switched by UV-A and blue light between two activity states, in good accordance with results of the previous spectroscopic studies on the Cop5 rhodopsin domain [17,18,19,20]. Thus, we name the chimera C1 “switch-Cyclop1” (and C2 “switch-Cyclop2”), for bi-stable or “switchable cyclase opsin.”
The published study on the rhodopsin part of Cop5 (HKR1) revealed two light-switchable isoforms, Rh-UV (with absorption maximum at 380 nm) and Rh-Bl (with absorption maximum at 487 nm), which were suggested to be thermally stable in the darkness. Both isoforms can be efficiently interconverted by UV-A and blue light, and both states (Rh-UV and Rh-Bl) were shown to be absolutely stable for at least 50 min [17]. We determined the stability of enzyme activity in the dark after UV-A stimulation. We initially observed a gradual decrease of GC activity during incubation for several hours (Additional file 1: Figure S2A). However, it turned out that under our in vitro reaction conditions, addition of fresh ATP and GTP after 4 h completely restored the initial enzymatic activity (Additional file 1: Figure S2A). When fresh GTP and ATP were supplied every hour, the enzyme activity was constant in the dark for at least 6 h after UV-A- activation (Additional file 1: Figure S2B).
Activity of switch-Cyclop1 is switched by weak light pulses
To determine the light conditions required for activation, we first applied UV-A light ranging from 0.6 to 9.6 μW/mm2 with a 30 s duration and then measured activity in dark for 2, 10, and 18 min. As shown in Fig. 4a, activity increased with the increasing light power. 9.6 μW/mm2 is near the saturation for a 30 s illumination. With 30 s illumination half-maximal activity was obtained with ~ 2.6 μW/mm2, corresponding to a K0.5 of 78 μJ/mm2 (= 78 J/m2).
At a given light intensity, the activation should also depend on the duration, as confirmed by UV-A illumination of low intensity (0.6 μW/mm2) with different time windows (Fig. 4b). The required time for 50% activation is about 1.9 min with 0.6 μW/mm2, which results in a K0.5 of 68 μJ/mm2 (= 68 J/m2). We therefore estimate the required energy density for half-maximal activation by 380 nm photons at 73 J/m2.
We further evaluated the blue light conditions required for inhibiting the UV-A-activated switch-Cyclop1. After activation with 30 s 9.6 μW/mm2 UV-A light, 30 s illumination with blue (473 nm) light ranging from 0.6 to 9.6 μW/mm2 was applied to inhibit the enzyme. As shown in Fig. 4c, 30 s 0.5 μW/mm2 blue light inhibits ~ 50% of active switch-Cyclop1, and 30 s 9.6 μW/mm2 can inhibit more than 90% of active switch-Cyclop1. When applying lower light power and longer times, we determined that 0.6 μW/mm2 inhibits ~ 50% of active switch-Cyclop with ~ 30 s illumination (Fig. 4d). These results yield a K0.5 for blue light-induced inhibition of 15 J/m2 and of 20 J/m2, respectively. The estimated energy density for half-maximal inhibition by 473 nm photons is therefore 18 J/m2.
Similar to Cr2c-Cyclop1, functionality of switch-Cyclop1 requires Mg2+ and ATP (Additional file 1: Figure S3). Mg2+ plays distinct roles in regulating nucleotidyl cyclase activity and ATP is required for auto-phosphorylation of the conserved histidine by the kinase domain. When replacing Mg2+ with Ca2+, the enzyme activity was highly decreased, similar to the blue-inhibited state (Additional file 1: Figure S3). Ca2+ therefore cannot replace Mg2+.
Switch-Cyclop1 can sense different light ratios
Our results confirm the bi-stable regulation of the Cop5 Rhodopsin domain (HKR1) by UV-A and blue (or green) light, when fused to a domain with GC activity. However, in nature, there is always a mixture of photons of different wavelengths. We therefore tested how switch-Cyclop1 reacts to mixtures of 380 nm and 505 nm light.
We used 1 μW/mm2 continuous UV-A light for activation and different intensities of 505 nm light were added in parallel experiments. The switch-Cyclop1 activity decreases with the increasing ratio of 505 nm light (Fig. 5a). switch-Cyclop1 is an artificial chimera but it utilizes the full rhodopsin domain of Cop5 for photo sensing. This suggests that the Cop5 rhodopsin (HKR1) can sense the cyan/UV-A ratio in the living alga, if the protein Cop5 is expressed and is functional (e.g. as a heterodimer).
Cop5 and Cr2c-Cyclop1 probably co-exist in C. reinhardtii, but their rhodopsin part shows quite different characteristics: Cop5 rhodopsin is bi-stable and Cr2c-Cyclop1 rhodopsin is light-inhibited. However, we found that UV-A light (at 1 μW/mm2) cannot activate or inhibit Cr2c-Cyclop1, as its activity in UV-A is not different to the activity in dark (Fig. 5b).