Nanosecond retinal structure changes in K-590 during the room-temperature bacteriorhodopsin photocycle: picosecond time-resolved coherent anti-stokes Raman spectroscopy.

Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.     

Assignment of the hydrogen-out-of-plane and -in-plane vibrations of the retinal chromophore in the K intermediate of pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes of the protein leading to activation of the cognate transducer protein (pHtrII). Elucidation of the initial photoreaction, formation of the K intermediate of ppR, is important for understanding the mechanism of storage of photon energy. We have reported the K minus ppR Fourier transform infrared (FTIR) spectra, including several vibrational bands of the retinal, the protein, and internal water molecules. It is interesting that more vibrational bands were observed in the hydrogen-out-of-plane (HOOP) region than for the light-driven proton pump, bacteriorhodopsin. This result implied that the steric constraints on the retinal chromophore in the binding pocket of ppR are distributed more widely upon formation of the initial intermediate. In this study, we assigned the HOOP and hydrogen-in-plane vibrations by means of low-temperature FTIR spectroscopy applied to ppR reconstituted with retinal deuterated at C7, C8, C10-C12, C14, and C15. As a result, the 966 (+)/971 (-) and 958 (+)/961 (-) cm(-1) bands were assigned to the C7=C8 and C11=C12 Au HOOP modes, respectively, suggesting that the structural changes spread to the middle part of the retinal. The positive bands at 1001, 994, 987, and 979 cm(-1) were assigned to the C15-HOOP vibrations of the K intermediate, whose frequencies are similar to those of the K(L) intermediate of bacteriorhodopsin trapped at 135 K. Another positive band at 864 cm(-1) was assigned to the C14-HOOP vibration. Relatively many positive bands of hydrogen-in-plane vibrations supported the wide distribution of structural changes of the retinal as well. These results imply that the light energy was stored mainly in the distortions around the Schiff base region while some part of the energy was transferred to the distal part of the retinal.     

Assignment of the vibrational modes of the chromophores of iodopsin and bathoiodopsin: low-temperature fourier transform infrared spectroscopy of 13C- and 2H-labeled iodopsins

To investigate the chromophore structures of iodopsin and its low-temperature photoproducts, we have assigned their vibrational bands in the Fourier transform infrared (FTIR) spectra using iodopsin samples that were reconstituted with a series of (13)C- and deuterium-labeled retinals. The analyses of the vibrational bands in the fingerprint and hydrogen-out-of-plane (HOOP) regions indicated that the structure of the chromophores in the iodopsin system differs near their centers from those in the rhodopsin system. Compared to rhodopsin, the chromophore of the batho intermediate of iodopsin is twisted in the C(12) to C(14) regions but is more planar around C(11) region. The large amount of twisting was reduced by removing the chloride ion from the iodopsin, suggesting that this twisting hinders the relaxation of the torsion near C(11) necessary for the transition to the lumi intermediate and thus results in the thermal reversion of the batho intermediate back to the iodopsin. From the analyses of the C=NH and C=ND stretching bands, we conclude that the displacement of the Schiff base region upon photoisomerization of the chromophore is restricted, as is the case for rhodopsin. These results indicated that iodopsin's chromophore has a unique structure near its center and that this difference is enhanced by the binding of chloride nearby.     

Resonance Raman analysis of the Pr and Pfr forms of phytochrome

Resonance Raman vibrational spectra of the Pr and Pfr forms of oat phytochrome have been obtained at room temperature. When Pr is converted to Pfr, new bands appear in the C = C and C = N stretching region at 1622, 1599, and 1552 cm-1, indicating that a major structural change of the chromophore has occurred. The Pr to Pfr conversion results in an 11 cm-1 lowering of the N-H rocking band from 1323 to 1312 cm-1. Normal mode calculations correlate this frequency drop with a Z----E isomerization about the C15 = C16 bond. A line at 803 cm-1 in Pr is replaced by an unusually intense mode at 814 cm-1 in Pfr. Calculations on model tetrapyrrole chromophores suggest that these low-wavenumber modes are hydrogen out-of-plane (HOOP) wagging vibrations of the bridging C15 methine hydrogen and that both the intensity and frequency of the C15 HOOP mode are sensitive to the geometry around the C14-C15 and C15 = C16 bonds. The large intensity of the 814-cm-1 mode in Pfr indicates that the chromophore is highly distorted from planarity around the C15 methine bridge. If the Pr----Pfr conversion does involve a C15 = C16 Z----E isomerization, then the intensity of the C15 HOOP mode in Pfr argues that the chromophore has an E,anti conformation. On the basis of a comparison with the vibrational calculations, the low frequency (803 cm-1) and the reduced intensity of the C15 HOOP mode in Pr suggest that the chromophore in Pr adopts the C15-Z,syn conformation.     

Resonance Raman studies of the HOOP modes in octopus bathorhodopsin with deuterium-labeled retinal chromophores

Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm-1 for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm-1 to C10H and C14H HOOP modes, and the band at 940 cm-1 to C11H = C12H Au-like HOOP mode. Except for a 26 cm-1 downward shift, the C11H = C12H Au-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C10H and C14H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C7H and C8H HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C11H = C12H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C11 = C12 double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C11H = C12H Au-like HOOP mode. The twisted nature of the chromophore, semiquantitatively discussed here, likely affects the lambda max of the chromophore and its enthalpy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early photocycle structural changes in a bacteriorhodopsin mutant engineered to transmit photosensory signals

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) function as a light-driven proton pump and a receptor for negative phototaxis in haloarchaeal membranes, respectively. SRII transmits light signals through changes in protein-protein interaction with its transducer HtrII. Recently, we converted BR by three mutations into a form capable of transmitting photosignals to HtrII to mediate phototaxis responses. The BR triple mutant (BR-T) provides an opportunity to identify structural changes necessary to activate HtrII by comparing light-induced infrared spectral changes of BR, BR-T, and SRII. The hydrogen out-of-plane (HOOP) vibrations of the BR-T were very similar to those of SRII, indicating that they are distributed more extensively along the retinal chromophore than in BR, as in SRII. On the other hand, the bands of the protein moiety in BR-T are similar to those of BR, indicating that they are not specific to photosensing. The alteration of the O-H stretching vibration of Thr-204 in SRII, which we had previously shown to be essential for signal relay to HtrII, occurs also in BR-T. In addition, 1670(+)/1664(-) cm(-1) bands attributable to a distorted alpha-helix were observed in BR-T in a HtrII-dependent manner, as is seen in SRII. Thus, we identified similarities and dissimilarities of BR-T to BR and SRII. The results suggest signaling function of the structural changes of the HOOP vibrations, the O-H stretching vibration of the Thr-215 residue, and a distorted alpha-helix for the signal generation. We also succeeded in measurements of L minus initial state spectra of BR-T, which are the first FTIR spectra of L intermediates among sensory rhodopsins.     

Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

Raman microscope studies on the primary photochemistry of vertebrate visual pigments with absorption maxima from 430 to 502 nm

Raman microscope vibrational spectra have been recorded from single photoreceptor cells frozen at 77 K. Spectra of photostationary steady-state mixtures of visual pigments and their primary photoproducts were obtained from toad red rods (lambda max 502 nm), angelfish rods (lambda max 500 nm), gecko blue rods (lambda max 467 nm), and bullfrog green rods (lambda max 430 nm). All four photoproducts have enhanced low-wavenumber Raman lines at approximately 850, 875, and 915 cm-1 and show the anomalous decoupling of the 11- and 12-hydrogen out-of-plane (HOOP) wagging vibrations, as is observed in the bovine primary photoproduct. The low-wavenumber lines are enhanced in the resonance Raman spectrum by conformational distortion, and the uncoupling of the 11- and 12-hydrogen wags is caused by additional protein perturbations. The similarity of the HOOP modes in all four photoproducts indicates that the protein perturbations that uncouple the 11- and 12-hydrogen wags and that enhance the HOOP modes are very similar. Thus, these perturbations of the photoproduct Raman spectrum cannot be caused by the same protein-chromophore interactions that are responsible for wavelength regulation in these pigments.     

Time-resolved resonance raman structural studies of the pB' intermediate in the photocycle of photoactive yellow protein

Time-resolved resonance Raman spectroscopy is used to obtain chromophore vibrational spectra of the pR, pB', and pB intermediates during the photocycle of photoactive yellow protein. In the pR spectrum, the C8-C9 stretching mode at 998 cm(-1) is approximately 60 cm(-1) lower than in the dark state, and the combination of C-O stretching and C7H=C8H bending at 1283 cm(-1) is insensitive to D2O substitution. These results indicate that pR has a deprotonated, cis chromophore structure and that the hydrogen bonding to the chromophore phenolate oxygen is preserved and strengthened in the early photoproduct. However, the intense C7H=C8H hydrogen out-of-plane (HOOP) mode at 979 cm(-1) suggests that the chromophore in pR is distorted at the vinyl and adjacent C8-C9 bonds. The formation of pB' involves chromophore protonation based on the protonation state marker at 1174 cm(-1) and on the sensitivity of the COH bending at 1148 cm(-1) as well as the combined C-OH stretching and C7H=C8H bending mode at 1252 cm(-1) to D2O substitution. The hydrogen out-of-plane Raman intensity at 985 cm(-1) significantly decreases in pB', suggesting that the pR-to-pB' transition is the stage where the stored photon energy is transferred from the distorted chromophore to the protein, producing a more relaxed pB' chromophore structure. The C=O stretching mode downshifts from 1660 to 1651 cm(-1) in the pB'-to-pB transition, indicating the reformation of a hydrogen bond to the carbonyl oxygen. Based on reported x-ray data, this suggests that the chromophore ring flips during the transition from pB' to pB. These results confirm the existence and importance of the pB' intermediate in photoactive yellow protein receptor activation.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

FTIR spectroscopy of the all-trans form of Anabaena sensory rhodopsin at 77 K: hydrogen bond of a water between the Schiff base and Asp75

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria, and is believed to function as a photosensor interacting with a 14 kDa soluble protein. Most of the residues in the retinal binding pocket are similar in ASR except proline 206, where the corresponding amino acid in other archaeal-type rhodopsins is highly conserved aspartate that constitutes the counterion complex of the positively charged protonated Schiff base. The recently determined X-ray crystallographic structure of ASR revealed a water molecule between the Schiff base and Asp75 [Vogeley, L., Sineshchekov, O. A., Trivedi, V. D., Sasaki, J., Spudich, J. L., and Luecke, H. (2004) Science 306, 1390-1393], as well as the case for bacteriorhodopsin (BR), a typical transport rhodopsin working as a proton pump. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all-trans form of ASR at 77 K, and compared the local structure around the chromophore and their structural changes upon retinal photoisomerization with those of BR. The K intermediate minus ASR difference spectra were essentially similar to those for BR, indicating that photoisomerization yields formation of the distorted 13-cis form. In contrast, little amide I bands were observed for ASR. The presence of the proline-specific vibrational bands suggests that peptide backbone alterations are limited to the Pro206 moiety in the K state of ASR. The N-D stretching of the Schiff base is presumably located at 2163 (-) and 2125 (-) cm(-)(1) in ASR, suggesting that the hydrogen bonding strength of the Schiff base in ASR is similar to that in BR. A remarkable difference between ASR and BR was revealed from water bands. Although ASR possesses a bridged water molecule like BR, the O-D stretching of water molecules was observed only in the >2500 cm(-)(1) region for ASR. We interpreted that the weak hydrogen bond of the bridged water between the Schiff base and Asp75 originates from their geometry. Since ASR does not pump protons, our result supports the working hypothesis that the existence of strongly hydrogen bonded water molecules is essential for proton pumping activity in archaeal rhodopsins.     

Structural change of threonine 89 upon photoisomerization in bacteriorhodopsin as revealed by polarized FTIR spectroscopy.

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.     

Structural changes in bacteriorhodopsin following retinal photoisomerization from the 13-cis form

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm(-)(1) for the K state of 13C-BR (13C-BR(K)) versus a vibration at 957 cm(-)(1) for the K state of AT-BR (AT-BR(K)). In AT-BR(K), but not in 13C-BR(K), the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR(K) were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BR(K). These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR(K), it is located elsewhere in 13C-BR(K). By use of [zeta-(15)N]lysine-labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D(2)O) at 2173 and 2056 cm(-)(1), close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm(-)(1) in 13C-BR(K) versus values of 2495 and 2468 cm(-)(1) for AT-BR(K), suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR(K) does not occur in 13C-BR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.     

Complete assignment of the hydrogen out-of-plane wagging vibrations of bathorhodopsin: chromophore structure and energy storage in the primary photoproduct of vision

Resonance Raman vibrational spectra of the retinal chromophore in bathorhodopsin have been obtained after regenerating bovine visual pigments with an extensive series of 13C- and deuterium-labeled retinals. A low-temperature spinning cell technique was used to produce high-quality bathorhodopsin spectra exhibiting resolved hydrogen out-of-plane wagging vibrations at 838, 850, 858, 875, and 921 cm-1. The isotopic shifts and a normal coordinate analysis permit the assignment of these lines to the HC7 = C8H Bg, C14H, C12H, C10H, and C11H hydrogen out-of-plane wagging modes, respectively. The coupling constant between the C11H and C12H wags as well as the C12H wag force constant are unusually low compared to those of retinal model compounds. This quantitatively confirms the lack of coupling between the C11H and C12H wags and the low C12H wag vibrational frequency noted earlier by Eyring et al. [(1982) Biochemistry 21, 384]. The force constants for the C10H and C14H wags are also significantly below the values observed in model compounds. We suggest that the perturbed hydrogen out-of-plane wagging and C-C stretching force constants for the C10-C11 = C12-C13 region of the chromophore in bathorhodopsin result from electrostatic interactions with a charged protein residue. This interaction may also contribute to the 33 kcal/mol energy storage in bathorhodopsin.     

Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy.

Time-resolved resonance Raman microchip flow experiments have been performed on the lumirhodopsin (Lumi) and metarhodopsin I (Meta I) photointermediates of rhodopsin at room temperature to elucidate the structure of the chromophore in each species as well as changes in protein-chromophore interactions. Transient Raman spectra of Lumi and Meta I with delay times of 16 micros and 1 ms, respectively, are obtained by using a microprobe system to focus displaced pump and probe laser beams in a microfabricated flow channel and to detect the scattering. The fingerprint modes of both species are very similar and characteristic of an all-trans chromophore. Lumi exhibits a relatively normal hydrogen-out-of-plane (HOOP) doublet at 951/959 cm(-1), while Meta I has a single HOOP band at 957 cm(-1). These results suggest that the transitions from bathorhodopsin to Lumi and Meta I involve a relaxation of the chromophore to a more planar all-trans conformation and the elimination of the structural perturbation that uncouples the 11H and 12H wags in bathorhodopsin. Surprisingly, the protonated Schiff base C=N stretching mode in Lumi (1638 cm(-1)) is unusually low compared to those in rhodopsin and bathorhodopsin, and the C=ND stretching mode shifts down by only 7 cm(-1) in D2O buffer. This indicates that the Schiff base hydrogen bonding is dramatically weakened in the bathorhodopsin to Lumi transition. However, the C=N stretching mode in Meta I is found at 1654 cm(-1) and exhibits a normal deuteration-induced downshift of 24 cm(-1), identical to that of the all-trans protonated Schiff base. The structural relaxation of the chromophore-protein complex in the bathorhodopsin to Lumi transition thus appears to drive the Schiff base group out of its hydrogen-bonded environment near Glu113, and the hydrogen bonding recovers to a normal solvated PSB value but presumably a different hydrogen bond acceptor with the formation of Meta I.     

Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.     

Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology

Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with fast data acquisition and high sensitivity has been developed for vibrational imaging of live cells. High three-dimensional (3D) resolution is achieved with two collinearly overlapped near infrared picosecond beams and a water objective with a high numerical aperture. Forward-detected CARS (F-CARS) and epi-detected CARS (E-CARS) images are recorded simultaneously. F-CARS is used for visualizing features comparable to or larger than the excitation wavelength, while E-CARS allows detection of smaller features with a high contrast. F-CARS and E-CARS images of live and unstained cells reveal details invisible in differential interference-contrast images. High-speed vibrational imaging of unstained cells undergoing mitosis and apoptosis has been carried out. For live NIH 3T3 cells in metaphase, 3D distribution of chromosomes is mapped at the frequency of the DNA backbone Raman band, while the vesicles surrounding the nucleus is imaged by E-CARS at the frequency of the C-H stretching Raman band. Apoptosis in NIH 3T3 cells is monitored using the CARS signal from aliphatic C-H stretching vibration.     

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vibrational spectroscopy of biopolymers under mechanical stress: processing cellulose spectra using bandshift difference integrals

Mechanical stretching of covalent bonds, for example when a fibrous polymer is loaded in tension, results in their stretching vibrational bands in the infrared or Raman spectrum being shifted to lower frequency. Conversely stretching a hydrogen bond shifts the stretching vibrational mode of the donor covalent X-H bond to higher frequency. These band shifts are small and difficult to detect in complex regions of the spectrum where differently affected bands overlap. This paper describes a method of integrating the difference spectra (spectrum under tensile strain minus spectrum at zero tensile strain) to recover the shape of the bands that are shifted and the spectral variation in bandshift. The application of this method to two sets of vibrational spectra of cellulose under tension is described. In one example, C-O-C stretching bands of highly crystalline tunicate cellulose were observed to shift to lower frequency under axial strain. In the other example, a group of overlapping O-D stretching bands in partially deuterated cellulose showed varied bandshifts under axial strain, some bandshifts being positive as expected due to extension of axially oriented hydrogen bonds while others were negative. The possibility of constructing spectral plots of bandshift has the potential to clarify the interpretation of overlapped, shifting bands in the vibrational spectra of polymers under tension.     

The photobleaching sequence of a short-wavelength visual pigment.

The photobleaching pathway of a short-wavelength cone opsin purified in delipidated form (lambda(max) = 425 nm) is reported. The batho intermediate of the violet cone opsin generated at 45 K has an absorption maximum at 450 nm. The batho intermediate thermally decays to the lumi intermediate (lambda(max) = 435 nm) at 200 K. The lumi intermediate decays to the meta I (lambda(max) = 420 nm) and meta II (lambda(max) = 388 nm) intermediates at 258 and 263 K, respectively. The meta II intermediate decays to free retinal and opsin at >270 K. At 45, 75, and 140 K, the photochemical excitation of the violet cone opsin at 425 nm generates the batho intermediate at high concentrations under moderate illumination. The batho intermediate spectra, generated via decomposing the photostationary state spectra at 45 and 140 K, are identical and have properties typical of batho intermediates of other visual pigments. Extended illumination of the violet cone opsin at 75 K, however, generates a red-shifted photostationary state (relative to both the dark and the batho intermediates) that has as absorption maximum at approximately 470 nm, and thermally reverts to form the normal batho intermediate when warmed to 140 K. We conclude that this red-shifted photostationary state is a metastable state, characterized by a higher-energy protein conformation that allows relaxation of the all-trans chromophore into a more planar conformation. FTIR spectroscopy of violet cone opsin indicates conclusively that the chromophore is protonated. A similar transformation of the rhodopsin binding site generates a model for the VCOP binding site that predicts roughly 75% of the observed blue shift of the violet cone pigment relative to rhodopsin. MNDO-PSDCI calculations indicate that secondary interactions involving the binding site residues are as important as the first-order chromophore protein interactions in mediating the wavelength maximum.