Picosecond Resonance Coherent Anti-Stokes Raman Spectroscopy of Light- and Dark-Adapted Bacteriorhodopsin

Coherent anti-Stokes Raman scattering (CARS) from the C=C stretching band region of retinal, recorded with low-power, 7-ps (fwhm) laser excitation selected to be in electronic resonance with the retinal chromophore, is presented for light- and dark-adapted bacteriorhodopsin (BR) samples. By minimizing photochemistry with low-power excitation, the picosecond resonance CARS (PR/CARS) spectrum of BR-570 (13-trans, 15-anti-retinal) is obtained directly from the light-adapted BR sample. The PR/CARS spectrum of BR-548 (13-cis, 15-syn retinal) is derived from a quantitative analysis of PR/CARS data from dark-adapted BR which contains comparable amounts of BR-570 and BR-548. Band origin positions, lineshapes, relative intensities, and phase factors describing the electronic resonances are obtained from a quantitative fit of the PR/CARS spectra to third-order susceptibility (χ⁽³⁾) relationships. These PR/CARS data, the first reported for a molecular system as large as BR, demonstrate the experimental viability of CARS for recording the vibrational spectrum of a chromophore within a trans-membrane protein. In addition, the χ⁽³⁾ analysis shows that a CARS spectrum of a mixture of isomeric chromophores can be quantitatively separated into CARS data assignable to its individual components. The significance of these results with respect to analyzing picosecond time-resolved CARS data from intermediates in the BR photocycle is discussed.     

Determination of the total unsaturation in oils and margarines by fourier transform raman spectroscopy

An improved Raman spectroscopic procedure for the determination of the total unsaturation in oils and fats using Fourier Transform Raman (FT-Raman) spectroscopy is described. An important advantage of FT-Raman for these samples is that the spectra are fluorescence-free unlike dispersive Raman which often uses visible excitation. Samples can be analyzed without any pre-treatment thus eliminating the need for dissolution in toxic solvents. The short acquisition time of FT-Raman and the ease of application allowed for a rapid sample turnover.     

Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP)

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.     

Application of FTIR Spectroscopy to the Investigation of Dark Structures and Photoreactions of Visual Pigments

Experiments are reviewed in which the photoreactions of the visual pigments rhodopsin and octopus rhodopsin have been investigated by FTIR spectroscopy. It is shown that from the spectra important characteristic structural elements can be derived for the dark state as well as for the intermediates. Since the spectra contain contributions from the chromophore as well as from the protein, methods are described which allow one to discriminate between them. In the case of rhodopsin, the influence of single amino acids on spectra and function is assessed by investigations of specific mutants. The role of the chromophore-protein interaction for the photoreaction and the activation mechanism is addressed by studies of modified rhodopsins containing retinal analogues as chromophores. Characteristics of the active state, which is capable of transducin activation, are especially emphasized.     

Spatially resolved near-infrared excited Raman spectroscopy of nanocrystalline diamond films

Scanning Raman spectroscopic measurements were performed on nanocrystalline diamond thin films with 0.5 μm lateral steps and excitation spot limited to 1 μm in diameter using 488 nm and 785 nm excitation wavelengths. The comparison of the spectra measured with different excitation energies showed that in contrast to the well-known five bands in the 488 nm excited Raman spectra of nanocrystalline diamond a number of narrow peaks appears in the spectra when using near-infrared excitation. The intensity and position of the latter vary when moving the excitation spot along the sample. The detailed analysis of the sequences showed that the 785 nm excited Raman spectroscopy allows the detection and identification of the Raman peaks arising from individual diamond crystallites of the nanocrystalline diamond films.     

Raman scattering characterization of CVD graphite films

Raman spectroscopic study has been performed for thin graphite films grown on nickel substrates by chemical vapor deposition from a mixture of hydrogen and methane activated by a direct current discharge. Depending on the growth conditions, the CVD films are composed of graphene layers parallel to the substrate surface or of plate-like crystallites with the predominant orientation of their graphene layers perpendicular to the substrate surface. A comparison of the Raman spectra for the CVD films and for the highly oriented pyrolytic graphite has been performed. The mechanisms governing the Raman scattering process in the films are discussed. An important role of a double resonance mechanism in the Raman spectra of these graphite-based materials has been revealed. The Raman band positions and intensities and their dependence on excitation wavelength confirm a high degree of the structural order in the CVD graphite films.     

蛋白质光学信息记录材料细菌视紫红质

光敏蛋白质细菌视紫红质(bR)存在于嗜盐菌紫膜中。bR分子吸收光子后,视黄醛构型发生变化,产生一系列中间态并经历一个独特的光循环过程,同时完成质子泵功能。bR具有高的空间分辨率、高的光灵敏度、高的循环次数及良好的热稳定性和非线性光学特性,是一种性能优良的生物光学材料,可广泛应用于空间光调制器、投影显示、全息记录、模式识别、光学信息存储等方面,在分子电子学、生物计算机等领域具有诱人的应用前景。     

Raman Spectral Features Associated with Chromophores in High-Yield Pulps

High-yield pulp chromophores are not easily analysed. In Raman spectroscopic studies, advantage can be taken of the fact that chromophores absorb visible light, and they are therefore expected to result in manifestation of the resonance Raman effect. In this effect, the Raman scattering coefficient depends upon the wavelength used to excite the spectrum. The contribution of chromophores in the spectra of mechanical pulps was identified by recording Raman spectra excited at two different wavelengths (514.5 and 647.1 nm). In these spectra, the intensities of certain bands and the band width of the 1595 cm^?1 band changed when the wavelength of excitation was changed. These changes were clear evidence of the presence of the chromophores and useful indicators of the extent to which the chromophores were present in the mechanical pulps. Spectral contributions due to chromophores were found at 1120, 1595, 1620, and 1654 cm^?1. Raman studies of bleached pulps were consistent with these observations and indicated that chromophores were not completely removed by the bleaching processes.     

Doping level change of polyaniline film during its electrochemical growth process

Polyaniline (PANI) films doped with hydrochloric acid have been electrochemically deposited on mirrorlike platinum electrode surfaces by a cyclic voltammogram method. The Raman spectra of doped PANI films were investigated by excitation with a 633‐ or 785‐nm laser beam. It was found that the overall features of the Raman spectra depend strongly on the film thickness, due mainly to that the doping level of PANI film increases during the film‐growth process. X‐ray photoelectron spectroscopic (XPS) analysis and ultraviolet (UV)‐visible absorption spectrum results also confirmed this finding. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 171–177, 2004     

Solution-state (15)N NMR spectroscopic study of alpha-C-phycocyanin: implications for the structure of the chromophore-binding pocket of the cyanobacterial phytochrome Cph1

The detailed structure of the chromophore-binding pocket in phytochrome proteins and the structural changes associated with its photocycle are still matters of debate. Insight into the structure and dynamics of the binding pocket has been gained through the comparison of a (15)N NMR spectrum of alpha-C-phycocyanin, which is often used as a model system for the study of phytochromes, with the previously described (15)N NMR spectrum of the cyanobacterial phytochrome Cph1. The former spectrum supports the hypothesis that all four nitrogen atoms of the alpha-C-phycocyanin chromophore are protonated, in analogy with the proposed protonation state for the P(r) and P(fr) forms of Cph1. The spectra show that the chromophores in both proteins exhibit a distinct dynamic behavior, as also indicated by a NOESY spectrum of Cph1. Finally, stereochemical arguments and a Cph1 homology model support the hypothesis that the chromophore in Cph1 is most likely in the ZZZssa conformation in the P(r) form of the protein.     

Acid-Base Equilibria and the Proton Pump in Bacteriorhodopsin

The light-induced proton pump in bacteriorhodopsin is reviewed with emphasis on acid-base equilibria of protein residues and of the retinal Schiff base moiety. Pump mechanisms in bR and in some of its mutants are classified in terms of light-induced pK_a changes (class I) or light-induced exposure changes, in which the proton accessibility of the protein changes from the outside to the inside of the membrane (class II). A discussion of the theoretical basis of the factors which determine the pK_a of ionizable protein groups is followed by a review of the experimental phenomena associated with the titration of residues in both unphotolyzed bR and during its photocycle. The time-resolved titrations of the Schiff base and of the Asp-85 residue are discussed in terms of the accessibility of the two groups to external protons. Finally, the molecular aspects of the pH-dependent proton pump in native bR and in various mutants are analyzed, focusing on the mechanism of the initial proton release reaction and on the subsequent molecular switch which allows reprotonation from the inside of the cell. Special attention is devoted to the question of coupling between the photocycle intermediates (primary M formation and decay) and the transmembrane proton translocation. Recent work with bR mutants raise the question as to whether proton transfer from the Schiff base to Asp-85 at the M stage is directly responsible for proton translocation, as well as for the reprotonation switch.     

Identification of mNeonGreen as a pH-Dependent,Turn-On Fluorescent Protein Sensor for Chloride

Chloride-sensitive fluorescent proteins generated from laboratory evolution have a characteristic tyrosine residue that interacts with a chloride ion and π-stacks with the chromophore. However, the engineered yellow-green fluorescent protein mNeonGreen lacks this interaction but still binds chloride, as seen in a recently reported crystal structure. Based on its unique coordination sphere, we were curious if chloride could influence the optical properties of mNeonGreen. Here, we present the structure-guided identification and spectroscopic characterization of mNeonGreen as a turn-on fluorescent protein sensor for chloride. Our results show that chloride binding lowers the chromophore pK_a and shifts the equilibrium away from the weakly fluorescent phenol form to the highly fluorescent phenolate form, resulting in a pH-dependent, turn-on fluorescence response. Moreover, through mutagenesis, we link this sensing mechanism to a non-coordinating residue in the chloride binding pocket. This discovery sets the stage to further engineer mNeonGreen as a new fluorescent protein-based tool for imaging cellular chloride.     

Vibrational Spectra of Ca3SiO5: Ultraviolet Laser Raman Spectroscopy at High Temperatures

The Raman spectra of Ca₃SiO₅ have been successfully obtained at temperatures up to 1353 K by a continuous-wave ultraviolet Raman spectroscopic system. The Raman spectra do not change significantly with an increase in temperature, despite triclinic ( T )→monoclinic ( M )→rhombohedral ( R ) transitions. The present result is described in terms of the parameter related to temperature variation at constant pressure, defined in a manner similar to the Gruneisen parameter. Raman bands located above 350 cm⁻¹ show smaller values of the parameter than lower frequency modes, suggesting that these higher frequency modes are assigned to SiO₄ internal modes.     

Transient decay studies of photophysical processes in aromatic polymers: 2. Investigation of intramolecular excimer formation in copolymers of 1-vinyl naphthalene and methyl acrylate

Transient decay studies of the copolymer system 1-vinyl naphthalene/methyl acrylate using pulsed laser excitation have shown that monomer and excimer decay rates are not described by dual exponential functions. Triple exponential decays are attributed to the existence of emission from two kinetically distinct monomer sites in addition to that of the excimer. Variation of intramolecular chromophore concentration in the copolymers allows adoption of a versatile series of extrapolation procedures to derive individual rate constants in the photophysical reaction scheme governing intramolecular excimer formation. Use of this procedure yields a rate parameter governing excimer formation in which the dependence upon local chromophore concentration within the polymer coil is removed to a degree not possible in studies of homopolymers alone.     

Subpicosecond solvent dynamics in charge-transfer transitions: challenges and opportunities in resonance Raman spectroscopy

Resonance Raman spectroscopy is an established tool for the determination of structure and dynamics in electronically excited states. In condensed-phase systems, Raman excitation profiles and electronic absorption spectra depend on changes in molecular geometry and solvation structure induced by electronic excitation. Recent studies of solvent isotope effects on resonance Raman intensities in charge-transfer excitations reveal solvent dynamics taking place on a subpicosecond time scale and vibrational mode-specific solute-solvent interactions. These discoveries present challenges to the current working theories for analysis of resonance Raman and absorption spectra.     

Resonant Raman spectroscopy on enriched C carbon nanotubes

Isotopically enriched single-wall carbon nanotubes with different ¹³C concentrations were investigated by resonant Raman spectroscopy. Linear reductions of the Raman frequencies with an increase of ¹³C concentration are observed for the different nanotube Raman modes, and the effect of the reduced mass variation of the isotope mixture on the phonon frequencies is described through a simple harmonic oscillator model. In addition to the frequency dependence, the Raman linewidths as a function of the ¹³C concentration were also investigated and an expression describing this is presented. We observe an increase in the G band linewidth, as the ¹³C:¹²C ratio approaches unity. Measurements with different excitation energies were performed and the frequency dispersions of the D and G′ bands with laser energy were observed to be the same for ¹²C and ¹³C nanotubes, suggesting no changes in the electronic structure after isotope enrichment. Through analysis of the radial breathing modes in the Raman spectra obtained with different excitation energies, a relation between these modes frequency and the ¹³C nanotubes diameter was also established.     

Application of FTIR Spectroscopy to the Structural Study on the Function of Bacteriorhodopsin

Difference FTIR spectroscopy of the photointermediates of bacteriorhodopsin is informative for changes in H-bonding, the protonation states, and the bond orientation of functional residues such as C=O, N-H, and O-H of the chromophore, protein residues, peptide bonds, and internal water molecules. The vibrational bands of the chromophore are found at frequencies similar to those observed by resonance Raman spectra. Moreover, FTIR gives clear results on the N— in-plane bending vibration and C₁₄–C₁₅ stretching vibrational modes, both of which are useful for the analysis of the structure of the chromophore. The protonation states and H-bonding changes of intramembrane protonated aspartic acid residues can be revealed only by FTIR spectroscopy, in combination with proper isotope labeling and site-directed mutagenesis. The results with Asp-96 and Asp-85 in the L, M, and N photointermediates were especially useful for understanding the proton transfer mechanism. Besides amino acid groups in the protein, peptide C=O vibrations were assigned to a specific bond by the use of site-directed isotope labeling. Water molecules which undergo structural changes upon photoreaction were attributed specifically to those interacting with particular protein residues by mutational studies. On the basis of these FTIR studies, the role of the L intermediate is emphasized as the key intermediate that creates the conditions for the proton transfer reaction from the Schiff base to Asp-85 and subsequent proton uptake reactions in the cytoplasmic domain.     

Covalent binding of peroxidized phospholipid to protein: III. Reaction of individual phospholipids with different proteins

Various peroxidized phospholipids were reacted with proteins under N₂. In all cases, phospholipid is bound covalently to the proteins whose molecular size is increased. Both the amount of bound phospholipid and the increase in molecular size of the protein depends on the nature of the phospholipid. Ultraviolet (UV) absorption of the proteins is increased in qualitatively similar ways. Their difference spectra, which show a gradual increase in absorption from 400 nm toward shorter wavelength, differ from that of malonaldehyde-protein complexes. The various complexes of proteins and peroxidized phospholipids have similar fluorescence spectra showing two excitation maxima at 310–320 nm and at 340–350 nm, respectively, and emission maximum at ca. 400 nm. This is different from both fluorescence spectra of malonaldehyde-protein complexes and fluorescence spectra reported for proteins after reaction with peroxidized polyunsaturated fatty acids. Amino groups of the proteins are consumed in the reaction with peroxidized phospholipids. Blocking the amino groups decreases the binding of phospholipid considerably. Besides amino groups, other structures of the protein molecule react with the peroxidized phospholipids. The similar features of UV absorption, fluorescence, decrease of amino groups, and covalently bound phospholipid phosphorus of the various complexes suggest that they are formed by common type of reactions. The reactions seem to be different from those generally believed important between peroxidized lipid and protein. Important reacting species are compounds other than malonaldehyde. Preliminary report of this work was presented at the ISF/AOCS World Congress, New York, 1980.     

Photoreactions of the Photointermediates of Bacteriorhodopsin

The photochemical reactions of the intermediates of the photochemical cycle of bacteriorhodopsin (bR) are reviewed. These reactions constitute photochemical control of the cycle and provide an independent approach for the investigation of the mechanism of light energy transduction in the purple membrane. The absorption of a light quantum by the K, L, or M intermediates converts them back to bR. These transformations interrupt the photocycle so that no proton transfer occurs after absorption of the second quantum. The action of blue light on the M intermediate causes structural changes of the chromophore, as a result of which the Schiff base is reprotonated from Asp-85, not from Asp-96 as in the usual thermal transition of M. The photoreactions of the L, M, N, and O intermediates lead to the formation of new photoproducts. Studies of the photoconversion of the intermediates can serve as an additional source of information on the nature of photoprocesses in bR: they reveal several conformers of K and bR at 90 K, different M states, two N intermediates, and provide direct evidence for the existence of a thermal back reaction from N to M. The study of the photoreactions of the J, K, L, M, N, and O intermediates is a promising method for elucidating the structures and roles of these states. Reversible photoconversions of bR and its photointermediates provide a basis for potential applications of bR in optical registration of information.