Resonance Raman study of the dark-adapted form of the purple membrane protein.

The resonance Raman spectrum of the dark-adapted form of the purple membrane protein (bacteriorhodopsin) has been obtained and is compared to the light-adapted pigment and model chromophore spectra. As in the light-adapted form, the chromophore-protein linkage is found to be a protonated Schiff base. Electron delocalization appears to play the dominant role in color regulation. The dark-adapted spectrum indicates a conformation closer to 13-cis than the light-adapted spectrum.

Structural heterogeneity of the various forms of apomyoglobin: implications for protein folding.

Temperature-induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surrounding do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native-like (tightly packed) and molten globule-like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two "nucleation" events whereby native-like contacts are formed. One of these events, which involves AGH "core" formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermodynamical properties as well as formation kinetics.

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.

A resonance Raman study of the C=C stretch modes in bovine and octopus visual pigments with isotopically labeled retinal chromophores.

Previous resonance Raman spectroscopic studies of bovine and octopus rhodopsin and bathorhodopsin in the C-C stretch fingerprint region have shown drastically different spectral patterns, which suggest different chromophore-protein interactions. We have extended our resonance Raman studies of bovine and octopus pigments to the C=C stretch region in order to reveal a more detailed picture about the difference in retinal-protein interactions between these two pigments. The C=C stretch motions of the protonated retinal Schiff base are strongly coupled to form highly delocalized ethylenic modes located in the 1500 to 1650 cm-1 spectral region. In order to decouple these vibrations, a series of 11,12-D2-labeled retinals, with additional 13C labeling at C8, C10, C11 and C14, respectively, are used to determine the difference of specific C=C stretch modes between bovine and octopus pigments. Our results show that the C9=C10 and C13=C14 stretch mode are about 20 cm-1 lower in the Raman spectrum of octopus bathorhodopsin than in bovine bathorhodopsin, while the other C=C stretch modes in these two bathorhodopsins are similar. In contrast, only the C9=C10 stretch mode in octopus rhodopsin is about 10 cm-1 lower than in bovine rhodopsin, while other C=C stretches are similar.

A study of the Schiff base mode in bovine rhodopsin and bathorhodopsin.

We have obtained the resonance Raman spectra of bovine rhodopsin, bathorhodopsin, and isorhodopsin for a series of isotopically labeled retinal chromophores. The specific substitutions are at retinal's protonated Schiff base moiety and include -HC = NH+-, -HC = ND+-, -H13C = NH+-, and -H13C = ND+-. Apart from the doubly labeled retinal, we find that the protonated Schiff base frequency is the same, within experimental error, for both rhodopsin and bathorhodopsin for all the substitutions measured here and elsewhere. We develop a force field that accurately fits the observed ethylenic (C = C) and protonated Schiff base stretching frequencies of rhodopsin and labeled derivatives. Using MINDO/3 quantum mechanical procedures, we investigate the response of this force field, and the ethylenic and Schiff base stretching frequencies, to the placement of charges close to retinal's Schiff base moiety. Specifically, we find that the Schiff base frequency should be measurably affected by a 3.0-4.5-A movement of a negatively charged counterion from the positively charged protonated Schiff base moiety. That there is no experimentally discernible difference in the Schiff base frequency between rhodopsin and bathorhodopsin suggests that models for the efficient conversion of light to chemical energy in the rhodopsin to bathorhodopsin photoconversion based solely on salt bridge separation of the protonated Schiff base and its counterion are probably incorrect. We discuss various alternative models and the role of electrostatics in the rhodopsin to bathorhodopsin primary process.

Primary photochemistry and photoisomerization of retinal at 77 degrees K in cattle and squid rhodopsins.

The relative quantum yields of the photoreactions Rhodopsin in equilibrium Bathorhodopsin in equilibrium Isorhodopsin over an extended wavelength region have been determined in cattle and squid rhodopsins at 77 degrees K. The quantum yields were found to be wavelength independent and unchanged for samples suspended in D2O. The rhodopsin-bathorhodopsin forward and backward quantum yields sum to larger than one. These results are consistent with the previous suggestion that the excited singlet potential of rhodopsin has a single minimum along the 11-12 torsional coordinate. The values of the quantum yields are important for evaluating dynamic models of the rhodopsin-bathorhodopsin transition. We conclude that equilibration in the common excited state afer excitation of rhodopsin, as previously suggested, does not occur. Models involving molecular excitation trajectories conserving torsional momenta and excited state to ground state surface crossings better fit the data, and a semiquantitative analysis is presented. Probabilities of surface crossings are calculated.

Photochemistry of rhodopsin and isorhodopsin investigated on a picosecond time scale.

Bovine rhodopsin and isorhodopsin were excited with a single 530-nm, 7-ps light pulse emitted by a mode-locked Nd 3+ glass laser at room temperature. Within 3 ps of excitation, absorbance changes due to formation of bathorhodopsin were observed. The difference spectra generated during and 100 ps after pulse excitation are presented. The data show that bathorhodopsin formation is completed within 3 ps for both the primary pigments and suggest that a single common bathorhodopsin is photochemically formed from both primary pigments. Our findings provide additional support for the cis-trans isomerization model of the primary event in vision. Additional absorption transients that were observed near 670 and 460 nm are discussed.

Resonance Raman study of the primary photochemistry of bacteriorhodopsin.

Resonance Raman multicomponent spectra of the light-adapted form of bacteriorhodopsin, bRLA568, and its first photoproduct, K628, have been obtained at liquid nitrogen temperatures. The spectra of both bRLA568 and K628 could be obtained with the known sample compositions under our irradiating conditions and computer subtraction techniques. In agreement with previous results, we find that both bRLA568 and K628 contain chromophores linked to the apoprotein by protonated Schiff bases of retinal. Neither pigment form, suspended in H2O or 2H2O, compares closely to the spectral features of all-trans and 13-cis protonated and deuterated model chromophores, respectively. The data are consistent with other results, suggesting that a chromophore isomerization takes place in the bRLA568-to-K628 phototransition. However, the exact structure of the in situ chromophore would appear not to involve simple trans-to-13-cis structures found in solution.

The core of apomyoglobin E-form folds at the diffusion limit.

The E-form of apomyoglobin has been characterized using infrared and fluorescence spectroscopies, revealing a compact core with native like contacts, most probably consisting of 15-20 residues of the A, G and H helices of apomyoglobin. Fast temperature-jump, time-resolved infrared measurements reveal that the core is formed within 96 micros at 46 degrees C, close to the diffusion limit for loop formation. Remarkably, the folding pathway of the E-form is such that the formation of a limited number of native-like contacts is not rate limiting, or that the contacts form on the same time scale expected for diffusion controlled loop formation.

Molecular flow resonance Raman effect from retinal and rhodopsin.

We have performed resonance enhanced Raman measurements of retinal isomers in solution (all-trans, 11-cis, 9-cis, and 13-cis) and cetyltrimethylammonium bromide (CTAB) detergent extracts of bovine rhodopsin near physiological temperatures (17 degrees C). In order to measure these photolabile systems, we have developed a general technique which allows Raman measurements of any photosensitive material. This technique involves imposing a molecular velocity transverse to the Raman exciting laser beam sufficient to ensure that any given molecule moves through the beam so that it has little probability of absorbing a photon. We have also measured the resonance Raman spectra of crystals of the same retinal isomers. The data show that each isomer has a distinct and characteristic Raman spectra and that the spectrum of 11-cis-retinal is quite similar but not identical with that of rhodopsin and similarly for 9-cis-retinal compared with isorhodopsin. In agreement with previous work, the Raman data demonstrate that retinal and opsin are joined by a protonated Schiff base. Due to the fact that the Raman spectra of 11-cis-retinal (solution) and rhodopsin show bands near 998 and 1018 cm(-1), a spectral region previously assigned to C-Me stretching motions, it is suggested that 11-cis-retinal in solution is compased of a mixture of 12-s-trans and 12-s-cis, and that the conformation of rhodopsin is (perhaps distorted) 12-s-trans.

Effects of various anions on the Raman spectrum of halorhodopsin.

Resonance Raman experiments were conducted to probe and understand the effect of various anions on halorhodopsin. These included monoatomic anions Cl- and Br-, which bind to the so-called halorhodopsin binding sites I and II, and polyatomic anions NO3- and ClO4-, which bind to site I only. The two types of ions clearly show different effects on the vibrational spectrum of the chromophore. The differences are not localized to the Schiff base region of the molecule, but extend to the chromophore structure-sensitive fingerprint region as well. We find that the protonated Schiff base frequency is at 1,633 cm-1 for Cl- and Br- ions, as reported previously for Cl-. However, we find that two Schiff base frequencies characterize halorhodopsin upon binding of the polyatomic anions. One frequency lies at the same location as that found for the monoatomic anions and the other is at 1,645 cm-1. Halorhodopsin with bound NO3- and ClO4- thus may consist of two heterogeneous structures in equilibrium. This heterogeneity does not seem to correlate with a retinal isomeric heterogeneity, which we can also demonstrate in these samples. The results suggest that anions binding to site I do not bind to the Schiff base directly, but can influence chromophore and/or protein conformational states.

The calf gamma crystallins--a Raman spectroscopic study.

The solution structures of the four major components of bovine lens gamma-crystallin, gamma s, gamma II, gamma III and gamma IV are compared using Raman spectroscopy. The spectral region sensitive to the vibrational frequencies of aromatic and sulfur containing residues and to the backbone skeletal stretching modes (500-1000 cm-1), and that reflecting secondary structure (1,000-1,700 cm-1) are strikingly similar in all four gamma-crystallin fractions. These similarities are indicative of the dominant anti-parallel beta sheet structure common to all the gamma-crystallins. A comparison of the ratios of the Raman intensities at 850 cm-1 and 830 cm-1 (I850/I830), an empirical measure of the degree of hydrogen bonding of phenolic hydroxyl groups, suggests that the tyrosine residues in all the gamma-crystallin fractions are moderately hydrogen bonded. Distinct differences in the solution structures of the gamma-crystallins were observed in the higher energy end of the vibrational Raman spectra. The sulfhydryl stretching frequencies for the gamma-crystallins exhibit complex splitting patterns in the 2,500-2,600 cm-1 region. These patterns are due to the competing effects of hydrogen bonding and S-pi interactions with neighboring aromatic residues. All five proteins exhibit multiple, but distinct, thiol frequencies, suggesting that the microenvironments of the cysteine residues in these proteins are significantly different.

Resonance Raman study of the pink membrane photochemically prepared from the deionized blue membrane of H. halobium.

We report here the Resonance Raman spectrum of a 'pink' membrane (lambda max approximately 495 nm) photochemically generated from the deionized 'blue' membrane (Chang et al., 1985). Comparison of the Raman spectrum of the pink membrane with that of the model compounds, as well as the chromophore extraction data, indicate that the chromophore in the pink membrane is in the 9-cis configuration. The Schiff base peak at approximately 1,652 cm-1 shifts to approximately 1,622 cm-1 upon deuteration of the pink membrane, showing that the chromophore is bound to the bacterio-opsin by a protonated Schiff base linkage. The location of the Schiff base peak, as well as the 30 cm-1 shift that it undergoes upon deuteration, are quite different from the corresponding values for the native bacteriorhodopsin, suggesting differences in the local environment for the Schiff base in these pigments.

Resonance raman spectroscopy of an ultraviolet-sensitive insect rhodopsin.

We present the first visual pigment resonance Raman spectra from the UV-sensitive eyes of an insect, Ascalaphus macaronius (owlfly). This pigment contains 11-cis-retinal as the chromophore. Raman data have been obtained for the acid metarhodopsin at 10 degrees C in both H2O and D2O. The C = N stretching mode at 1660 cm-1 in H2O shifts to 1631 cm-1 upon deuteriation of the sample, clearly showing a protonated Schiff base linkage between the chromophore and the protein. The structure-sensitive fingerprint region shows similarities to the all-trans-protonated Schiff base of model retinal chromophores, as well as to the octopus acid metarhodopsin and bovine metarhodopsin I. Although spectra measured at -100 degrees C with 406.7-nm excitation, to enhance scattering from rhodopsin (lambda max 345 nm), contain a significant contribution from a small amount of contaminants [cytochrome(s) and/or accessory pigment] in the sample, the C = N stretch at 1664 cm-1 suggests a protonated Schiff base linkage between the chromophore and the protein in rhodopsin as well. For comparison, this mode also appears at approximately 1660 cm-1 in both the vertebrate (bovine) and the invertebrate (octopus) rhodopsins. These data are particularly interesting since the absorption maximum of 345 nm for rhodopsin might be expected to originate from an unprotonated Schiff base linkage. That the Schiff base linkage in the owlfly rhodopsin, like in bovine and in octopus, is protonated suggests that a charged chromophore is essential to visual transduction.

Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin.

We report the fast relaxation dynamics of "native" apomyoglobin (pH 5.3) following a 10-ns, laser-induced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales (nu = 1633 cm(-1),tau = 48 ns; nu = 1650 cm(-1),tau = 132 micros). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm(-1)) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm(-1)). Folding rate coefficients inferred from the observed relaxations at 60 degrees C are k(f)(solvated) = (7 to 20) x 10(6) s(-1) and k(f)(native) = 3.6 x 10(3) s(-1), respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60 degrees C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.