Properties of sensory rhodopsins and the receptortransducer complex

An important prerequisite for the analysis of the structural and biophysical properties of photoreceptors and their transducers is their availability. Originally, sensors were prepared from their natural host [96-99], However, due to their low cellular concentration - in wild-type H. salinarum there are only 2000-3000 copies of SRI [100] - molecular genetic tools had to be applied. A homologous expression system was introduced that allowed the overexpression of SRI [101-103] as well as the heterologous expression of NpSRII [104]. A decisive step forward was taken when it became possible to functionally express NpSRII in E. coli [105], This method proved to be successful not only for the facile preparation of NpSRII and HR [106] but also for SRI [107] using a His-tag as an affinity label. In a similar way a truncated form of NpHtrll could also be prepared [108,109],

1.3.1 Primary sequences

The functional and structural properties of sensory rhodopsins are determined by their primary structures. A two-dimensional structural map of NpSRII at 6.9 Á [110] and more recently high-resolution structures are available. Already the two-dimensional map clearly demonstrates the structural similarity between NpSRII and BR, which had already been deduced from an alignment of the corresponding primary sequences [36-38,111]. The highest percentage of homology is found at those sites which constitute the retinal binding pocket (sequences of-30 homologous rhodopsins have been published so far [112]). This is also true for an archaeal rhodopsin-like pigment (NOP-1) detected from Neurospora crassa [113,114], the first example of an archaeal rhodopsin discovered outside its own kingdom. Interestingly, according to its amino acid sequence it is more similar to BR than to SRI or HsSRII (e.g. the position of Asp96 (BR) is conserved); however, its photochemistry resembles that of sensory rhodopsins [114] (see Figure 2 for a sequence alignment of the four archaetypical rhodopsins BR, HR, SRI, and HsSRII). Most recently archaeal rhodopsins have also been discovered in marine microbial populations. Apparently, these pigments (named proteorhodopsins) function as light-driven proton pumps involved in phototrophy [115,116].

Comparing primary sequences of sensors, proton pumps, and halide pumps, obvious differences are connected to proton release and to the proton uptake channels (Figure 4). In BR the key residues are Asp85 and Asp96, which are crucially involved in the proton pump mechanism. After light excitation the Schiff base proton is transferred to Asp85, with concomitant release of a proton into the extracellular buffer. Once the salt bridge between the proto-nated Schiff base and the negatively charged Asp85 has been broken in the so-called M-state a protein switch can occur, which alters the accessibility of the Schiff base from the extracellular channel towards the cytoplasmic channel. Thus, the Schiff base can be reprotonated from the cytoplasmic side via Asp96, thereby completing the vectorial proton transfer across the membrane (see Lanyi for a detailed discussion of proton transfer reactions in BR [8]). In SRs the Asp 96 is replaced by an aromatic residue, thus interfering with an optimal proton transfer from the cytoplasm to the Schiff base (see below). A scheme of the proton transfer steps, comparing BR and NpSRII, is depicted in Figure 4.

Figure 4. Comparison of proton transfer steps in BR (left) and NpSRII (right). After light excitation the proton from the Schiff base is, during the L^M transition, transferred to an Asp residue (Asp85; Asp75). The time course and the mechanism of the susequent steps are different for BR and NpSRII. Whereas in BR Asp96 donates its proton to the Schiff base, this reaction is not possible for NpSRII (as well as for SRI and HsSRII) because an aromatic residue (F86) has replaced Asp96. Instead the reprotonation has to occur directly from the cytoplasm. The proton release to the extracellular medium is also different for the two pigments. Whereas BR releases the proton during the L-^M transition, in NpSRII this only happens in the last step of the photocycle (O—>NpSRII). The proton in the circle depicts sites connected to a hydrogen-bonded network with an excess proton. Abbreviations: c-channel, cytoplasmic channel; e-channel, extracellular channel.

Figure 4. Comparison of proton transfer steps in BR (left) and NpSRII (right). After light excitation the proton from the Schiff base is, during the L^M transition, transferred to an Asp residue (Asp85; Asp75). The time course and the mechanism of the susequent steps are different for BR and NpSRII. Whereas in BR Asp96 donates its proton to the Schiff base, this reaction is not possible for NpSRII (as well as for SRI and HsSRII) because an aromatic residue (F86) has replaced Asp96. Instead the reprotonation has to occur directly from the cytoplasm. The proton release to the extracellular medium is also different for the two pigments. Whereas BR releases the proton during the L-^M transition, in NpSRII this only happens in the last step of the photocycle (O—>NpSRII). The proton in the circle depicts sites connected to a hydrogen-bonded network with an excess proton. Abbreviations: c-channel, cytoplasmic channel; e-channel, extracellular channel.

1.3.2 Absorption spectra

All rhodopsins, if excited with light corresponding to their absorption maxima, display a characteristic photocycle. These maxima are for BR, HR and SRI above 560 nm. An exception is observed for HsSRII, which absorbs maximally at 490 nm (the homologous protein from N. pharaonis has a maximum at 500 nm; see Figure 5).

The reason for this reduced opsin shift is not yet clear. The opsin shift is a measure (in cm-1) of the protein's influence on the chromophore absorption maximum. The colour regulation in BR has been explained by a synergistic effect of a 6-s-trans bond at the B-ionone ring (as is found in SRI [117]) together with a complex counterion at the protonated Schiff base [118]. Experiments with retinal analogues indicated that in HsSRII and/or NpSRII the retinal binding site is more restricted than in BR [119]. A planarisation

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