Conformational Changes Detected in a Sensory Rhodopsin II-Transducer Complex

Abstract

Sensory rhodopsins (SRs) are light receptors that belong to the growing family of microbial rhodopsins. SRs have now been found in all three major domains of life including archaea, bacteria, and eukaryotes. One of the most extensively studied sensory rhodopsins is SRII, which controls a blue light avoidance motility response in the halophilic archaeon Natronobacterium pharaonis. This seven-helix integral membrane protein forms a tight intermolecular complex with its cognate transducer protein, HtrII. In this work, the structural changes occurring in a fusion complex consisting of SRII and the two transmembrane helices (TM1 and TM2) of HtrII were investigated by time-resolved Fourier transform infrared difference spectroscopy. Although most of the structural changes observed in SRII are conserved in the fusion complex, several distinct changes are found. A reduction in the intensity of a prominent amide I band observed for SRII indicates that its structural changes are altered in the fusion complex, possibly because of the close interaction of TM2 with the F helix, which interferes with the F helix outward tilt. Deprotonation of at least one Asp/Glu residue is detected in the transducer-free receptor with a pK a near 7 that is abolished or altered in the fusion complex. Changes are also detected in spectral regions characteristic of Asn and Tyr vibrations. At high hydration levels, transducer-fusion interactions lead to a stabilization of an M-like intermediate that most likely corresponds to an active signaling form of the transducer. These findings are discussed in the context of a recently elucidated x-ray structure of the fusion complex. Sensory rhodopsin (SR)1 II is a seven-helix integral membrane protein found in halophilic archaea that functions as a light receptor for negative phototaxis (1). Together with sensory rhodopsin I (SRI), which is a dual photoattractant/photorepellent receptor (2); bacteriorhodopsin (BR), a light-driven proton pump; and halorhodopsin, a light-driven chloride pump, these proteins belong to a widespread family of photoactive retinylidene proteins or microbial rhodopsins (3). Microbial rhodopsins have several common features including an alltrans-retinylidene chromophore covalently attached to the protein via a protonated Schiff base linkage. Light absorption results in an all-trans 3 13-cis-isomerization of the chromophore that triggers subsequent conformational changes associated with a photocycle characteristic of each protein. Members of the microbial rhodopsin family have recently been found in diverse organisms including marine proteobacteria (4), cyanobacteria (5), and lower eukaryotes such as Neurospora crassa (6) and Chlamydomonas reinhardtii (7).Sensory rhodopsin II transmits the photorepellent signal to the cell cytoplasm by activating the transmembrane domain of its cognate transducer HtrII (8), 2 which is tightly bound to the receptor helices F and G (9). The photocycle of SRII comprises several photointermediates (10 -13), including the blue-shifted M and red-shifte...

Photoactivation Perturbs the Membrane-embedded Contacts between Sensory Rhodopsin II and Its Transducer

“…2). This effect is similar to the difference observed previously between the spectra of wild-type SRII-HtrII complex and free receptor (27). In the spectra of tyrosine model compounds, a characteristic band appears between 1255 and 1275 cm Ϫ1 due to the C-O stretching mode 7Јa (CO) of a tyrosine side chain (31), whose frequency is sensitive to the hydrogen-bonding environment (32 occurs during formation of M (see "Discussion").…”

“…It is also absent in both mutant spectra. As noted previously (27), its frequency is typical for the CϭO stretching mode of an asparagine side chain. This vibration is found near 1678 cm Ϫ1 in the model compound spectra and depending on the hydrogen bonding environment can shift significantly, with the lower frequency corresponding to a stronger hydrogen bond (33 .…”

“…2) near 1694 cm Ϫ1 was previously identified in the SRIIHtrII complex but not in the receptor alone (27). It is also absent in both mutant spectra.…”

“…FTIR Difference Spectroscopy-The sample preparation and FTIR measurement procedures have been described previously (27). Rapidscan time-resolved FTIR spectroscopy was performed using a BRUKER IFS 66 v/s FTIR spectrometer (Bruker Optics, Billerica, MA) operating at 4 cm Ϫ1 spectral resolution and 240 kHz scanner velocity corresponding to a data acquisition window of 18 ms.…”

Active State of Sensory Rhodopsin II: Structural Determinants for Signal Transfer and Proton Pumping

“…EPR experiments also show that reordering of helix F and TM2 occurs, believed to represent the signal, occurs concomitantly with O-decay 6 . FTIR studies [30][31][32] indicate, that the more active state is present in the crystal, the larger are the diffraction resolution losses. Therefore, optimum conditions had to be established, at which both the active state occupancy and the resolution would be reasonable.…”