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. 2011 May 4;100(9):2275–2282. doi: 10.1016/j.bpj.2011.03.017

The Signal Transfer from the Receptor NpSRII to the Transducer NpHtrII Is Not Hampered by the D75N Mutation

Julia Holterhues †, Enrica Bordignon †, Daniel Klose †, Christian Rickert †, Johann P Klare †, Swetlana Martell ‡, Lin Li ‡, Martin Engelhard ‡, Heinz-Jürgen Steinhoff †,∗
PMCID: PMC3149259  PMID: 21539797

Abstract

Sensory rhodopsin II (NpSRII) is a phototaxis receptor of Natronomonas pharaonis that performs its function in complex with its cognate transducer (NpHtrII). Upon light activation NpSRII triggers by means of NpHtrII a signal transduction chain homologous to the two component system in eubacterial chemotaxis. The D75N mutant of NpSRII, which lacks the blue-shifted M intermediate and therefore exhibits a significantly faster photocycle compared to the wild-type, mediates normal phototaxis responses demonstrating that deprotonation of the Schiff base is not a prerequisite for transducer activation. Using site-directed spin labeling and time resolved electron paramagnetic-resonance spectroscopy, we show that the mechanism revealed for activation of the wild-type complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transmembrane transducer helix TM2, is also valid for the D75N variant. Apparently, the D75N mutation shifts the ground state conformation of NpSRII-D75N and its cognate transducer into the direction of the signaling state.

Introduction

In Natronomonas pharaonis and Halobacterium salinarum four different membrane-embedded retinylidene proteins, namely the ion pumps bacteriorhodopsin (BR) and halorhodopsin (HR), and the mediators of phototaxis, sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII, also named phoborhodopsin) have been identified. An additional proton transporter and a new sensory rhodopsin (SRM) were found in Haloarcula marismortui (1). Similar structural architectures consist of seven transmembrane helices (A–G) with the retinal chromophore covalently bound to a conserved lysine residue on helix G via a Schiff base linkage (for recent reviews see (2–5)). Furthermore, all retinylidene proteins exhibit similar photocycles upon light activation (2).

The two photosensors, SRI and SRII, enable the bacteria to seek favorable light conditions for the function of BR and HR or to avoid photooxidative stress. SRI displays a dual functionality in response to blue and orange light. H. salinarum is attracted by orange light, and additional near-UV light triggers a photophobic response (6). H. salinarum is also repelled by green-blue light (7)), and SRII, which is expressed with an absorption maximum around 500 nm in both H. salinarum and N. pharaonis is responsible for the green-blue light photophobic answer of the cells (8–10).

Both sensory proteins transmit the light-generated signal to tightly bound transducer proteins, HtrI and HtrII, which are homologous to eubacterial chemotaxis receptors, such as aspartate and serine receptors of Escherichia coli (Tar and Tsr, respectively (11–13)). The signaling complex exhibits a 2:2 stoichiometry with a transducer dimer flanked by two receptor molecules. The interaction surface in the protein complex consists of helices F and G of the sensory rhodopsin and the two transmembrane helices, TM1 and TM2, of the transducer (14,15) (see Fig. 1 A).

Figure 1.

Figure 1

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(A) Top view from the cytoplasm and (B) side view perpendicular to the bilayer normal of the 2:2 complex (pdb: 1H2S (14)) of NpSRII and NpHtrII. The spheres depict the Cβ atoms of the spin-labeled residues. Aspartate 75 and the retinal molecule are highlighted in stick representation. (C) Zoom of the retinal binding pocket and the four water molecules (numbered spheres) in the hydrogen network comprising D75. The picture was generated with visual molecular dynamics (37).

Absorption of a photon by the archaeal rhodopsins leads to isomerization of all-trans to 13-cis retinal, initiating a sequence of intermediates with distinct optical and/or structural properties (2). These archetypical intermediates (named K, L, M, N, and O), originally assigned for the BR photocycle, have been also identified for sensory rhodopsin II from N. pharaonis (NpSRII) (16). This reaction cycle is characterized by the following key events: Upon retinal isomerization, the protonated Schiff base deprotonates by transferring the proton to the Schiff base counterion D75 (D85 in BR). Subsequently, structural rearrangements lead to an outward tilt motion of the cytoplasmic moiety of helix F followed by reprotonation of the Schiff base and reformation of the receptor ground state resulting in retinal reisomerization to all-trans (reviewed in Klare et al. (2)). Previously, it was shown that activation of the transducer is not directly coupled to the M state formation of NpSRII characterized by the proton transfer from the Schiff base to D75 (17). Using site-directed spin labeling and electron paramagnetic resonance (EPR) (15,17,18), it was found that with the transition from the early M state (M1) to the late M state (M2) a structural rearrangement occurs in NpSRII, analogous to the helix F motion observed for BR (19–21). This rearrangement in turn causes a rotation and/or displacement of the transducer helix TM2, which can be considered as the starting event for the signal transduction (15,22).

In the D75N mutant of NpSRII the Asp side chain representing the counterion for the protonated retinal Schiff base is replaced by Asn thereby abolishing its function as a proton acceptor. Consequently, a blue-shifted M state is missing and only long-wavelength intermediates are observed with a photocycle turnover ∼50 times faster than that of the wild-type (23,24). Interestingly, conformational changes as detected by Fourier transform infrared (FTIR) spectroscopy are quite similar for both wild-type and D75N mutant (23). It is therefore not surprising that the D75N mutation does not abolish the photophobic response of the cells (25). These results show that deprotonation of the Schiff base and formation of the M state is no prerequisite for transducer activation. Hence, helix F motion should also be observable for D75N.

To address this question, we employed site-directed spin labeling in combination with time-resolved EPR spectroscopy. In analogy to previous investigations on wild-type NpSRII (26) (in the following the spin-labeled NpSRII variants are also denoted wild-type to distinguish them from the spin-labeled D75N mutants), we labeled positions on helices C, F, and G of NpSRII-D75N and investigated the EPR spectral changes induced by light excitation of the receptor protein. In addition, transducer molecules bound to NpSRII-D75N and spin labeled at position 78, which has been shown to be an appropriate site to probe conformational changes within the transducer dimer (15), were investigated to see if the signaling mechanism revealed for the NpSRII/NpHtrII complex is also valid for the D75N receptor variant (spin-labeled sites are depicted in Fig. 1 B). The cumulated results presented in this study are in line with light-induced conformational rearrangements taking place at the cytoplasmic side of NpSRII-D75N consisting of an outward tilt motion of helix F.

Materials and Methods

Protein expression and spin labeling

C-terminal His-tag protein expressions, spin labeling with the MTS spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate, and sample preparation were performed according to (15,18,27). (The spin label side chain is designated R1 in the following.) The NpSRII/NpHtrII complex was formed with transducer constructs truncated at position 157 (NpHtrII157).

Flash photolysis

The light from a 50 W halogen lamp was filtered with infrared-cutoff and a 520 nm interference filter and passed through the sample cuvette placed in a temperature-controlled sample holder. The transmitted light was passed through a monochromator and detected with a photodiode. A flashlight (flash duration 80 μs) or a laser (SpitLight 400, InnoLas Laser GmbH, Krailing, Germany, combined with an OPO basiScan120, GWU-Lasertechnik, Erftstadt, Germany, pulse duration 6–8 ns, 500 nm, 2 mJ) was used for light excitation. The amplified signal was recorded with an analog-to-digital converter connected to a standard PC. The transient absorption changes were detected at 298 K for NpSRII-D75N/NpHtrII157 complexes reconstituted in purple membrane lipids at a concentration of ∼0.27 mg mL−1. For the transitions between the late photocycle intermediates (t > 0.2 ms) studied here, the kinetics determined by flashlight excitation were indistinguishable from that determined by laser light pulse excitation.

Continuous wave EPR

Room temperature EPR spectra were recorded with a homemade EPR spectrometer equipped with a dielectric resonator (Bruker Biospin, Rheinstetten, Germany). The microwave power was set to 1.0 mW, the B-field modulation amplitude was 0.15 mT. A volume of 15 μL of sample at concentration of 15–20 mg/mL was loaded into glass capillaries (0.9 mm inner diameter).

Low temperature (160 K) spectra were recorded with a homemade EPR spectrometer equipped with an AEG H103 rectangular cavity. A volume of 40 μL of sample at final concentrations of 15–20 mg/mL was loaded into EPR quartz capillaries (3 mm inner diameter). The microwave power was set to 0.2 mW, the B-field modulation amplitude was 0.25 mT. The magnetic field was measured with a Bruker B-NM 12 B-field meter and the temperature was stabilized with a continuous flow cryostat (Oxford ESR 900).

Time-resolved EPR

Time-resolved EPR measurements were performed as described previously (26). For transient EPR experiments with the NpSRII-D75N mutant, an excimer pumped dye laser (Lambda Physik, Göttingen, Germany) was tuned to 530 nm with the laser dye Coumarin 153 or 520 nm with Coumarin 307. The energy per light pulse amounted to between 1.2 and 1.5 mJ for 20 ns pulse duration. EPR transient traces were recorded for 3–4 s after the laser flash at fixed B-field positions and sampled at a rate of 104 s−1. To obtain a proper signal/noise ratio between 103 and 104 EPR transient traces were averaged. The integration time of the lock-in amplifier (Signal recovery, Wokingham, United Kingdom, model 7225) was set to 5 ms for the detection of the kinetics of the EPR signal changes, allowing the recording of the EPR signal decay after photoexcitation. The corresponding EPR signal decay time constants were obtained by fitting the experimental EPR transient traces with mono- or biexponential functions. Difference spectra were obtained according to the method described previously (26).

Results

To monitor light-induced structural rearrangements in the complex NpSRII-D75N/NpHtrII, sites for spin labeling were selected according to those previously chosen to study helix F movements in the NpSRII/NpHtrII wild-type complex (26). Three sites are located close to the cytoplasmic surface of helices C, F, and G (L89C, L159F, L213G; the superscripts denote the corresponding helix). The fourth site is located at the interface between the two TM2 helices of the two transducer molecules in the 2:2 complex (V78TM2). Fig. 1 shows the location of the Cβ atoms of the selected positions in the x-ray structure of the membrane region of the 2:2 complex (pdb: 1H2S (14)).

The EPR-based analysis was performed on NpSRII-D75N in complex with the transducer NpHtrII truncated at position 157 (NpHtrII157). This membrane domain of the transducer has previously been shown to be a good model for studies of the receptor/transducer interaction (28,29). All samples were reconstituted into purple membrane lipids.

Spin label side chain mobility

To analyze possible effects of the D75N mutation on the conformation of the receptor, EPR spectra were recorded for NpSRII-D75N L89R1C, L159R1F, and L213R1G and overlaid with previously published spectra obtained for the wild-type receptor (18,26) (see Fig. 2). The spectral linewidth and the apparent hyperfine splitting reflect the mobility of the spin label side chain. High mobility is characterized by small linewidths and small apparent hyperfine splitting due to the averaging of the anisotropic components of the g and hyperfine tensors. Restriction of the nitroxide motion by secondary or tertiary interactions is revealed by larger linewidths and hyperfine splitting. For all spin label side chains investigated the reorientational motion is strongly restricted. This behavior is expected for positions 159F and 213G, which are oriented into the interior of the protein. The spin label side chain bound to position 89C, although pointing outward is immobilized as well, as already observed for this site in the wild-type protein (26). This indicates strong interaction of the R1 side chain with residues in the same or a neighboring receptor, or with the headgroups of the lipid bilayer. Double electron-electron resonance measurements on D75N/L89R1C revealed interspin distances of 2.1 and 4.1 nm proving the close packing of receptors in the membrane.

Figure 2.

Figure 2

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(A) Room temperature EPR spectra of the spin labels bound to the indicated positions in sensory rhodopsin II and the transducer normalized to maximum amplitude. The wild-type NpSRII (gray) and the mutant NpSRII-D75N (black) were measured in complex with the transducer NpHtrII157 and reconstituted in purple membrane lipids. (B) Room temperature EPR spectra for NpSRII/NpHtrII-V78R1 and NpSRII-D75N/NpHtrII-V78R1 reconstituted in purple membrane lipids normalized to the same number of spins. In some of the spectra shown in (A) and (B) a negligible amount of unbound spin label (sharp lines) is present that could not be eliminated by repetitive washing.

The spectrum of D75N/L213R1G reveals a small but significant increase in mobility compared to the wild-type protein. This is evidence for a perturbation of interhelical connectivity by the D75N mutation.

Light-induced structural changes in NpSRII-D75N

Fig. 3 A shows transient light-dark difference amplitudes at selected B-field values superimposed on the EPR spectra of the dark state and on EPR difference spectra (288 K–298 K for NpSRII-D75N/L89R1C and 298 K–288 K for NpSRII-D75N/L159R1F). The insets present the corresponding spectra and difference amplitudes previously obtained for the wild-type receptor under the same experimental conditions (26). For all spin-labeled positions considerable light-induced spectral changes could be detected. Apparently, the loss of the Schiff base proton acceptor does not prevent light-induced conformational changes to occur.

Figure 3.

Figure 3

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(A) Transient difference amplitudes (light–dark) of the NpSRII-D75N/NpHtrII157 mutants. The sticks represent the maximum amplitude (multiplied by 250) of the light-induced EPR signal changes detected at the corresponding B-field position. For comparison, the corresponding transient difference spectra obtained with the wild-type receptor under the same experimental conditions are shown in the insets (26). EPR spectra and temperature difference spectra (gray) for D75N/L89R1C (288 K–298 K, multiplied by 2.5) and D75N/L159R1F (298 K–288 K, multiplied by 4) detected at 0.3 mT modulation amplitude are superimposed. (B) EPR transients (black, NpSRII-D75N; continuous gray line, wild-type, data taken from (26)) recorded at selected B-field positions corresponding to the maximum of the transient EPR signals and optical transients for NpSRII-D75N measured at 520 nm (broken gray line). Due to the limited bandwidth of the EPR lock-in detection (time constant 5 ms) spectral changes for times exceeding 30 ms (20 ms in (C)) are shown. (C) Same as in (A) and (B) for NpSRII-D75N/NpHtrII157-V78R1TM2.

Comparison of the transient difference amplitudes detected in the complex consisting of the wild-type or D75N receptor reveals site-specific effects. To understand the difference spectra in terms of increase and decrease of the spin label mobility, we evaluated the fingerprint region close to the extremes of the center line. Nonzero difference amplitudes close to the maximum and close to the minimum of the center line are due to a change of the spectral line width, which may be due to changes of the R1 side chain mobility or of the spin-spin interaction. Center peak amplitude changes due to changes of the environmental polarity of the spin label have minor effects (26). To discriminate between mobility and interspin distance changes, the transient light-dark difference amplitudes are qualitatively compared with experimental temperature difference spectra (298 K–288 K or 288 K–298 K), which are assumed to mainly reflect changes in spin label mobility, and with the difference of spectra that were simulated with different reorientational correlation times (21 ns–17 ns, see Fig. 3 B in (26)).

The transient light-dark difference amplitudes for D75N/L159R1F and for the corresponding wild-type sample are in qualitative agreement with the experimental temperature difference spectrum (298 K–288 K) and with the difference of the simulated spectra (26). In particular, the agreement of the characteristic sign reversals in the B-field regions of the center peaks observed for the transient amplitudes and for the temperature difference spectrum is strong evidence for a transient mobilization of the R1 side chain upon light excitation. Conformational changes at this site can be correlated to the outward tilt of the cytoplasmic moiety of helix F in the late M state for the wild-type receptor (15,17,18,26). Obviously, a similar conformational change occurs during the photocycle of the D75N mutant that lacks the M state.

Compared to D75N/L159R1F the transient changes in the center field region for D75N/L89R1C are reversed. In addition, the behavior of the transient light-dark difference amplitudes for D75N/L89R1C is not identical to that of the wild-type. The sign reversals, characteristic for mobility changes as present in the temperature difference spectrum, is obvious for the wild-type data but not for the data of D75N/L89R1C. Instead, the shape of the light-dark difference amplitudes resembles that of an inverted absorption spectrum. This may be evidence for a transient broadening due to the increase of spin-spin interaction in addition to a possible transient immobilization of the spin label side chain. In fact, double electron-electron resonance measurements of D75N/L89R1C show that the distance to the next spin label side chain in the sensory rhodopsin-transducer cluster yields 2.1 nm. This close proximity was not observed for the wild-type, which might be due to a concentration dependent different packing in the membrane. A light-induced rearrangement of the sensory rhodopsin-transducer cluster may decrease the interspin distance and correspondingly leads to increased spin-spin interaction.

For D75N/L213R1G light-induced changes could only be detected at B-field positions close to the spectral extremes. Remarkably, the inversion of the difference amplitude with respect to the wild-type receptor indicates inverse changes in the mobility upon light excitation.

The kinetic characterization of the photocycle provides the basis for the interpretation of the time-resolved EPR results in terms of the photocycle intermediates. Furthermore, the integrity of the structure and function of the spin-labeled NpSRII-D75N cysteine mutants can be characterized by analyzing the absorbance changes in the visible spectrum. Fig. 3 B shows a selection of transient EPR traces detected at the extremes of the EPR difference spectra in the NpSRII-D75N and wild-type receptor under the same experimental conditions in comparison with the light-induced absorbance changes recorded at 520 nm. Generally, the decay of the optical and EPR transient traces detected for NpSRII-D75N variants is more than one order of magnitude faster than those found for the wild-type variants due to the lack of intermediate M. Starting at 1 ms after the light flash excitation, a sum of two exponentials is sufficient to fit the optical absorbance changes. The time constant values are given in Table 1, together with the relative amplitudes of the respective exponentials. A comparison reveals that the time constants of the transducer-free NpSRII-D75N (24), of NpSRII-D75N/NpHtrII157-V78R1 and NpSRII-D75N/L89R1C/NpHtrII157 are similar, whereas the decay time constants for NpSRII-D75N/L159R1F and NpSRII-D75N/L213R1G are increased up to fivefold compared to the NpSRII-D75N. Thus, modifications of these positions in helices F and G influence the photocycle of NpSRII-D75N to a higher extent than was found for wild-type variants, where spin labeling of these sites has a much smaller influence (26).

Table 1.

Time constants of the recovery of the initial state of NpSRII-D75N and NpSRII-D75N/NpHtrII as determined by time-resolved EPR and optical absorption (520 nm) spectroscopy

NpSRII-D75N∗ L89R1C/NpHtrII157 L159R1F/NpHtrII157 L213R1G/NpHtrII157 V78R1TM2NpHtrII157
τ520nm/ms 7 8 ± 1 (76%) 12 ± 1 (72%) 26 ± 1 (71%) 6 ± 1 (80%)
28 44 ± 2 (24%) 64 ± 5 (28%) 132 ± 10 (29%) 24 ± 2 (20%)
τEPR/ms – 40 ± 20 50 ± 20 100 ± 40 45 ± 20
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EPR and optical time constants were determined by mono- or biexponential fitting of the traces shown in Fig. 3 with the relative amplitudes of the respective exponentials given in parentheses.

∗

Data taken from Schmies et al. (24).

The decays of the EPR signals follow the transient absorption changes detected at the ground state absorption maximum (520 nm for NpSRII-D75N) (24), revealing that the structural changes observed decay in phase with the optically detected recovery of NpSRII-D75N to the initial state. The time constants of the EPR transient signal decays agree within experimental error with the larger time constants of the biexponential decays determined for the optical time traces of the respective NpSRII-D75N variants (cf. Table 1).

The signal transfer from NpSRII to NpHtrII157

The results show that the photocycle turnover of NpSRII-D75N is faster than that of the wild-type NpSRII corroborating earlier results determined by absorption- and FTIR spectroscopy (23,24). Furthermore, our results reveal that NpSRII-D75N undergoes light-induced conformational changes in the cytoplasmic region that differ from those observed for the wild-type. On the other hand, phototaxis is not impaired by the D75N mutation (25). Therefore, the signal transfer to the transducer should follow similar pathways as in the wild-type.

To verify this assumption, a receptor/transducer complex carrying the D75N mutation in the receptor and the V78R1 mutation in the transducer NpHtrII157 was studied. The spin label bound to position 78 is located at the interface between the two TM2 helices in the membrane region of the transducer (see Fig. 1). This site turned out to be an appropriate reporter of the TM2 rotation upon signal transfer, with an increase in interspin distance of 0.2 nm upon light activation (15).

Fig. 2 B shows the EPR spectra of NpSRII/NpHtrII157-V78R1TM2 and NpSRII-D75N/NpHtrII157-V78R1TM2. The spectra of both complexes exhibit strong restrictions of the nitroxide reorientational motion. Interestingly, the comparison of the spectra reveals a more pronounced line broadening for NpSRII/NpHtrII157-V78R1TM2, indicating a stronger spin-spin interaction of the transducers in complex with the wild-type receptor. This observation was confirmed by analysis of EPR spectra detected at 160 K, revealing a slightly larger interspin distance between R1 side chains at positions 78 in NpSRII-D75N/NpHtrII157-V78R1TM2 (1.5 ± 0.2 nm) compared to NpSRII/NpHtrII157-V78R1TM2 (1.3 ± 0.2 nm).

In Fig. 3 C (left) the EPR transient light-dark difference amplitudes are depicted. The signals for NpSRII-D75N/NpHtrII157-V78R1TM2 were lower than those obtained for the NpSRII/NpHtrII157-V78R1TM2 complex, which might be explained by already partially rotated TM2 helices in the ground state of the complex, in accordance with the larger interspin distance observed. The phase of the signal is in line with an increase of the interspin distance, at which a transient increase of the R1 side chain mobility cannot be excluded, similar to the results found for NpSRII/NpHtrII157-V78R1TM2. The time course of the transient EPR signal (Fig. 3 C, right) confirms that the light-induced conformational changes of the receptor induce conformational rearrangements of the transmembrane part of the transducer. The recovery of the initial state of the transducer in the NpSRII-D75N/NpHtrII157-V78R1TM2 complex is accelerated compared to the NpSRII/NpHtrII157-V78R1TM2 complex in line with the faster photocycle of NpSRII-D75N.

Discussion

The activation of NpSRII to a state, which transfers the signal to its cognate transducer, is initiated by the isomerization of the retinal that leads to an outward tilt of helix F during a spectrally silent transition from M1 to M2. This sequence of events has been substantiated by FTIR spectroscopy (23,30), biochemical techniques (31), and site-directed spin labeling EPR (15,17,18,26). The central idea of the helix F movement being the trigger for transducer activation was originally proposed by Spudich (30) who considered conformational changes observed in BR. The question arose whether the deprotonation of the Schiff base is a necessary step for helix F motion. There are several lines of evidence that such a proton transfer is not required. First, FTIR data (23) and laser-induced transient grating analysis (32) on NpSRII-D75N showed that backbone conformational changes are similar to those observed for wild-type NpSRII. Second, photocycle data obtained from microbial rhodopsins measured under various conditions revealed that conformational changes are not necessarily kinetically coupled to proton transfer reactions (2), as proposed previously by the isomerization/switch/transfer model (33). Finally, H. salinarum expressing NpSRII-D75N/NpHtrII is phototactically active (25).

In this study, we provide further evidence that NpSRII-D75N is functionally competent to activate NpHtrII, although the acceptor of the Schiff base proton is not present. The strongest indication for helix F motion is the light-induced transient mobilization of L159R1F, located in the interface between helices F and G, showing a separation of these two helices upon activation (26), although the extent of the helix F motion might be different compared to that in the wild-type complex. Further differences observed between NpSRII-D75N and the wild-type receptor concern the mobility of the L213R1 side chain and the shapes of the transient difference spectra for L89R1 and L213R1. The latter finding might be interpreted in the context of a loosened contact between helices C and G due to an altered hydrogen bond network in the vicinity of the Schiff base (see Fig. 1 C). Whereas in the wild-type the hydrogen-bond mediated connection between helices C and G is disturbed during formation of the M state (22), in NpSRII-D75N it is either already weakened or even not existent. Consequently, a conformational change as observed for helix G in the wild-type cannot be expected in the absence of the Schiff base proton acceptor group.

In conclusion, the EPR data are in line with the notion that a light-induced displacement of helix F is also preserved in NpSRII-D75N. According to the current model of receptor-transducer signal transfer this movement triggers a transient rotation of TM2. Indeed, light activation of the NpSRII-D75N/NpHtrII complex leads to EPR spectral changes of V78R1TM2 similar to those observed for the wild-type complex, although less pronounced. The reason for the latter observations can be derived from the present data: Because the receptor is most likely biased toward partial activation in the ground state as seen by weakening of the C-G interaction, the transducer dimer conformation should also be affected, which is indeed reflected in the larger interspin distance between the R1 side chains of NpHtrII-V78R1TM2 in the ground state. Therefore, receptor and transducer are excited from a preactivated state and, consequently, the conformational changes observed in both molecules during the transition to the fully activated state are less expressed compared to those of the wild-type complex. Nevertheless, the D75N mutant and the D75Q variant in complex with NpHtrII have been reported to display no constitutive activity but to be fully active in phototaxis (25). Interestingly, this study also revealed that NpSRII-D75Q in complex with the corresponding transducer protein from H. salinarum (HsHtrII) exhibits constitutive activity at low level and almost no phototactic activity. On the other hand, the analogous D73N mutant of H. salinarum SRII (HsSRII) is constitutively active but still able to respond to light activation (34), whereas HsSRII-D73Q combined with NpHtrII or HsHtrII behaves like the NpSRII-D75Q-HsHtrII combination. These observations, together with our data, suggest that neutralization of the Schiff base counterion and disruption of the hydrogen bond network lead to a shift of the ground state conformation of both sensory rhodopsins into the direction of the signaling state. Similar observations have also been made for the mutations of the Schiff base counterion in BR, D85. For BR-D85N, e.g., conformational changes including helices F and G have been observed (35), and the crystal structure of BR-D85S revealed an O-like photocycle intermediate (36). Given this shift of the conformational equilibrium toward the activated state of the retinal protein phototactic activity still depends on the type of transducer present. Thus, NpHtrII and HsHtrII seem to have different switching sensitivities. According to our results NpHtrII with NpSRII-D75N is in a preactivated state, which means that the dark state of the transducer exhibits an altered conformation. Nevertheless, this does not influence its phototactic behavior. On the other hand, the data by Sasaki et al.(25) show that for the corresponding complex from H. salinarum the transducer is already biased toward the activated state in the dark. Furthermore, this seems to be the case in the chimeric complexes, but there the signaling is obviously severely disturbed by the mutation because, in contrast to the wild-type sensors, no further activation by light can be observed. Further studies are needed to unravel this complex behavior, but the results presented here can already serve as a basis for understanding the mechanistic details of transducer activation.

Finally, despite a significantly shorter lifetime of the activated state in D75N a strong phototactic response is observed. Inoue et al. (32) proposed that the decays of the conformational changes in the receptor and in the transducer are uncoupled, leading to a significantly prolonged activation of the transducer compared to the short-lived D75N active state. A similar mechanism had already been suggested for the wild-type complex (15,17). However, the behavior of optical and EPR transients observed for the recoveries of the initial state of NpSRII-D75N (cf. Fig. 3 C) and of the TM2 initial orientation does not provide any evidence for their uncoupling. Nevertheless, conformational rearrangements of the HAMP domains and of the cytoplasmic domain of the transducer might be decoupled from the NpSRII initial state recovery. This will be subject of further detailed studies of the kinetics of receptor/transducer activation and deactivation.

Using site-directed spin labeling and time-resolved EPR spectroscopy, we have shown that the mechanism proposed for the activation of the NpSRII/NpHtrII complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transducer helix TM2, is likely to be valid also for the NpSRII-D75N/NpHtrII variant.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 431/P18 to J.P.K. and H.J.S., EN87/14-2 to M.E.) and the Max Planck Society (to M.E.).

Footnotes

Enrica Bordignon's present address is Laboratorium für Physikalische Chemie, ETH Zürich, Zürich, Switzerland.

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