Site-Directed Isotope Labeling and ATR-FTIR Difference Spectroscopy of Bacteriorhodopsin: The Peptide Carbonyl Group of Tyr 185 Is Structurally Active During the bR .fwdarw. N Transition

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Bacteriorhodopsin is a light-driven proton pump, which undergoes a photocycle consisting of several distinct intermediates. Previous studies have established that the M 3 N step of this photocycle involves a major conformational change of membrane embedded ␣-helices. In order to further investigate this conformational change, we have studied the photocycle of the high pH form of the mutant Asp-85 3 Asn (D85N Bacteriorhodopsin (bR)1 is a M r 26,000 protein found in the purple membrane of Halobacterium salinarium (1, 2). Although detailed information exists about the bR structure (3) and the protonation changes which occur at specific amino acid residues (4 -9), key questions still remain about the detailed mechanism of light-driven proton transport from the cytoplasmic to the extracellular medium. Perhaps the most fundamental question concerns understanding how the light-induced isomerization of the retinal chromophore couples to protein conformational changes. A closely related question applies to sensory rhodopsin I (10) and rhodopsin (11), whose signaling mechanisms also depend on the triggering of protein conformational changes by retinal isomerization.Several recent FTIR studies indicate that a major conformational change involving membrane embedded ␣-helices occurs in the bR structure during the M 3 N transition (12-17). For example, an intense negative band near 1669 cm Ϫ1 appears in the amide I region of the time-resolved FTIR difference spectrum of bR during formation of the N intermediate (13)(14)(15) and disappears during N decay (15,17). Similar results are also obtained from low temperature steady-state measurements, where the N intermediate is stabilized by either high pH or low temperature (16,18). In a recent study combining attenuated total reflection (ATR) FTIR difference spectroscopy and sitedirected isotope labeling (SDIL), it was found that part of the M 3 N transition involves the Tyr-185/Pro-186 region of the F-helix (19). This region may function as a hinge which allows reorientation of the F-helix. An interesting possibility is that this structural change is responsible for a switch in the Schiff base accessibility from an extracellular to a cytoplasmic proton conducting pathway, termed the E and C conformations (20,21). In general, such a switch has been postulated to be present in a variety of active transport pumps (21)(22)(23)(24)(25).In this report, we have used FTIR difference and resonance Raman spectroscopy to study the photoreactions of the high pH form of the Asp-85 3 Asn mutant (D85N alk ). Several earlier studies have established that the Schiff base of D85N deprotonates at high pH forming a new state with a max near 410 nm (20, 26 -29). Surprisingly, this deprotonated Schiff base species is still able to pump protons under blue light illumination in the same direction as wild type bR (30 -32). Our results show that the photocycle of the light-adapted form of D85N alk involves a conformational change of the protein, which is very similar to that occurring in wild type bR, des...

Section: Bacteriorhodopsin (Br)supporting

confidence: 70%

Protein Conformational Changes during the Bacteriorhodopsin Photocycle

Nilsson

Rath

Olejnik

et al. 1995

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“…Further progress should be possible by combining FTIR with methods of band assignment including isotope labeling and site-directed mutagenesis. In addition, it should be possible to apply specialized FTIR techniques including polarization (40,58) and attenuated total reflection (52) to probe changes in orientation of specific groups under well defined aqueous environment.…”

Section: Discussionmentioning

confidence: 99%

Conformational Changes Detected in a Sensory Rhodopsin II-Transducer Complex

Bergo

Spudich

et al. 2003

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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...

“…Earlier studies based on tyrosine isotope labels (41), site-directed mutagenesis (21), and site-directed isotope labeling (58) concluded that tyrosine bands identified in the FTIR difference spectra of bacteriorhodopsin arose from protonation changes in Tyr-185 located in the F-helix. Subsequent FTIR measurements on bR containing a 13 C isotope label in the C1 position of Tyr-185 indicated that the Tyr-185/Pro-186 peptide bond buried in the center of the F-helix undergoes some type of structural rearrangement (59). An interesting possibility is that the Tyr-185/Pro-186 bond serves as a hinge, which gives rise to the apparent tilting of the F-helix late in the bR photocycle (60,61).…”

Section: Figurementioning

confidence: 99%

Tyrosine Structural Changes Detected during the Photoactivation of Rhodopsin

DeLange¹,

Klaassen²,

Wallace-Williams³

et al. 1998

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We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho 3 MII difference spectra were obtained at 10°C for unlabeled recombinant Rho and isotope-labeled L-[ring-2 H 4 ]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2 H 4 ]Tyr and isolated using a His 6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR 3 M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the Fhelix, may form a hinge that facilitates ␣-helix movement.Elucidation of the mechanism of photoactivation of rhodopsin (Rho), 1 the light receptor in vision, remains an important problem in biology (1). Rhodopsin is an integral membrane protein found in the disc photoreceptor membranes of rod outer segments (2, 3) with a core structure consisting of seven transmembrane ␣-helices (4 -7). Upon light absorption, the rhodopsin chromophore, 11-cis-retinal, rapidly isomerizes to an alltrans configuration (8, 9) followed by a series of thermal transitions (Batho 3 Lumi 3 Meta I 3 Meta II) (10, 11). Signal transduction occurs upon formation of the Meta II intermediate, which binds and activates the G-protein transducin (12, 13). Because rhodopsin is a G-protein-coupled receptor, elucidation of its molecular mechanism is likely to be of general importance for the vast suprafamily of G-protein-coupled receptors, which include the ␤-adrenergic receptor (14, 15) and olfactory receptors (16).Thus far, bR is the only IMP with a seven-helix transmembrane motif whose structure has been elucidated at atomic resolution (17)(18)(19). This structure confirmed key features of an earlier "spectroscopic" model based in part on site-directed mutagenesis and FTIR difference spectroscopy (20 -23). For example, a retinal binding pocket was predicted on the basis of FTIR, UV-visible spectroscopy, and site-directed mutagenesis (20 -24), which is formed in part from several residues on the F-helix (helix 6 in rhodopsin), including two tryptophans (Trp-182, Trp-189), a proline (Pro-186), and a tyrosine (Tyr-185). These residues, along with several others from the C-helix (Trp-86, Thr-89, and Asp-85) are in close proximity to the retinal chromophore and act to constrain its structure in an all-trans configuration. In addition, these residues are in a good position to couple retinal isomerization to protein changes involved in proton transport, including a change in the structure and orientation of the F-helix.A comparable combination of Trp, Pro, and Tyr residues (WXPY) is fully conserved in helix 6 of all ...