Surface-enhanced resonance Raman scattering spectroscopy of bacterial photosynthetic membranes. The carotenoid of Rhodospirillum rubrum

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.     

Localization of carotenoids in plasma low-density lipoproteins studied by surface-enhanced resonance Raman spectroscopy

Surface-enhanced resonance Raman scattering (SERRS) spectra were measured for the beta-carotene and lycopene carotenoids present in low-density lipoproteins (LDLs), which were isolated from human plasma and adsorbed on roughened silver surfaces. The silver surface was modified by formation of a self-assembled monolayer (SAM) of carboxylate-terminated linear alkanethiols in order to simulate the LDL binding region of the cellular LDL receptor. Thiols of different chain length were used to produce SAMs of varying thicknesses. It was shown that carotenoids are not released from the LDL particle upon adsorption onto the bare and thiol modified silver surfaces. The SERRS studies indicated that beta-carotene and lycopene were present in the shell of the LDL particle. The dependence of SERRS on the distance from the silver surface was different for beta-carotene and lycopene in LDL. This observation suggests that the two carotenoids are located in different places of the LDL particle.     

In vivo states and functions of carotenoids in an aerobic photosynthetic bacterium,Erythrobacter longus

In vivo states and functions of carotenoids in the membranes and the isolated RC-B865 pigment-protein complexes from an aerobic photosynthetic bacterium, Erythrobacter longus, are investigated by means of fluorescence excitation and resonance Raman (RR) spectra. Erythroxanthin sulfate, a dominant carotenoid species in the membranes (>70%), is found not to transfer the absorbed light energy to bacteriochlorophyll (Bchl), and its RR spectra are similar between the in vivo and in vitro states. These observations indicate that erythroxanthin sulfate does not interact with either Bchl or proteins in the membranes, and suggest that its function may be limited to photoprotection by quenching the harmful singlet oxygen. On the other hand, two other carotenoid species contained in the isolated RC-B865 complexes, zeaxanthin and bacteriorubixanthinal, have a high efficiency of energy transfer to Bchl (88±5%). The RR spectra of these two carotenoids, each of which can be selectively obtained by choosing the excitation wavelength, show some characteristics of interactions with proteins or Bchl.Abbreviations Bchl bacteriochlorophyll a - FWHM full width at half maximum - PAGE polyacrylamide gel electrophoresis - RC reaction center - RR resonance Raman - SDS sodium dodecyl sulfate     

Nanoparticle assembly for sensitive DNA detection using SERRS

SERRS (surface-enhanced resonance Raman scattering) is a vibrational technique, whereby a relatively weak Raman scattering effect is enhanced through the use of a visible chromophore and a roughened metal surface. The direct analysis of DNA by SERRS requires the modification of a nucleic acid sequence to incorporate a chromophore, and adsorption of the modified sequence on to a roughened metal surface. Aggregated metallic nanoparticles are commonly used in the analysis of dye-labelled DNA by SERRS, allowing for detection levels that rival those gained from standard fluorescence-based techniques. In the present paper, we report on how SERRS can be exploited for the analysis of clinically relevant DNA samples. We also report on the ability of nanoparticles to aggregate as the result of a biologically significant event, as opposed to the use of an external charge-modifying agent. The self-assembly of metallic nanoparticles is shown to be a promising new technique in the move towards extremely sensitive methods of DNA analysis by SERRS.     

Surface enhanced resonance Raman scattering (SERRS) as a probe of the structural differences between the Pr and Pfr forms of phytochrome

Surface enhanced resonance Raman scattering (SERRS) spectra have been obtained from the active, far-red light absorbing (Pfr) and biologically inactive (Pr) forms of phytochrome adsorbed on silver colloids. Substantial differences between the SERRS spectra of the two forms in the low and high wavenumber regions are observed using 406.7 nm wavelength excitation. These differences reinforce those seen with 413.1 nm wavelength excitation in the high wavenumber region. Simultaneously, extensive differences are observed in the SERRS obtained from the same form in the low wavenumber region using 406.7 nm, as compared with 413.1 nm wavelength excitation. The relative intensity differences observed for the two forms, and those obtained using two slightly different excitation wavelengths to illuminate the same form, suggest that some type of subtle, protein-controlled structural variation is responsible for the spectroscopic differences. AZ----E isomerization during the Pr----Pfr phototransformation is consistent with the SERRS data, although the overall chromophore conformations are most likely conserved for the native Pr- and Pfr-phytochrome species. Slight out-of-plane ring twisting, accompanying the Pr----Pfr photoisomerization, may be responsible for the large difference in the spectroscopic properties of the native Pr and Pfr chromophores.     

Effect of nitric oxide on viscosity of nerve cell membranes

The influence of nitric oxide on the microviscosity of nerve cell membranes was investigated by resonance Raman (RR) spectroscopy. Changes in membrane viscosity were estimated from the resonance Raman-spectra of carotenoids localized in the axon plasmatic membrane and membranes of subcellular vesicles (cytosomes). For the nerve fibre, the extracellular addition of nitric oxide donor, sodium nitroprusside (0.5 mM), caused an increase in the 1526 cm(-1) band relative half-width and the modification of 1160 cm-1 band structure. Moreover, sodium nitroprusside led to an increase in the I1526/I1160 ratio by 13% in 25 min and a decrease in this ratio by 10% in 50 min. In the case of cytosomes, sodium nitroprusside (0.5 mM) resulted in the reduction of the I1526/I1160 ratio by 8% in 25 and 50 min. It was shown that the neuron rhythmic activity correlated with the I1526/I1160 ratio and cytosome membrane microviscosity. We suppose that nitric oxide causes a conformational transition of carotenoids in the axon plasmatic membrane and the membranes of cytosomes. This process can be due to nitric oxide-induced changes of the membrane microviscosity or potential.     

Semiempirical and Raman spectroscopic studies of carotenoids

Semiempirical AM1 calculations have been carried out for beta-carotene and the three xanthophylls (zeaxanthin, canthaxanthin, and astaxanthin) containing oxygen functions (hydroxy/keto groups) found in the majority of natural pigment. The fully optimized geometries correspond well with the X-ray structures of beta-carotene and canthaxanthin and indicate that substitutions on the terminal rings have a minimal effect on the conformation of the chromophore. Twisting along the polyenic chain results from steric interaction involving methyl substituents, and a Ci point group can be proposed for the four investigated carotenoids. AM1 calculated excitation energies for the strongly allowed excited states can be compared to with the experimental absorption band in the visible region, considering solvent effect. Resonance Raman (RR) and Fourier transform (FT) Raman spectra of natural astaxanthin as well as astaxanthins specifically 13C labeled at the positions 12,12'; 13,13'; 14,14'; 15,15'; 15, and 20,20' were recorded. Furthermore the RR and FT Raman spectra of the asymmetric carotenoid 20-norastaxanthin are presented. The data reveal a substantial amount of information about the coupling between the different vibrations, and enabled an extensive experimental verification of the theoretical normal-coordinate analysis previously performed on polyenic molecules [J Raman Spectrosc 1983, 14, 310-321; Advances in Infrared and Raman Spectroscopy, Vol. 12, 1985, pp. 115-178; Spectrochim Acta 1996, 53, 381-392; Biochim Biophys Acta 1994, 1185, 188-196]. The results make up a very interesting dataset which allowed the interpretation and/or observation of several, hitherto never observed or not well understood, effects in the Raman spectra of the differently labeled astaxanthins.     

Orientation of succinate dehydrogenase and cytochrome b558 in the Bacillus subtilis cytoplasmic membrane.

The orientation of the three subunits of the membrane-bound succinate dehydrogenase (SDH)-cytochrome b558 complex in Bacillus subtilis was studied in protoplasts ("right side out") and isolated membranes (random orientation), using immunoadsorption and surface labeling with [35S]diazobenzenesulfonate. Anti-SDH antibodies were adsorbed by isolated membranes but not by protoplasts. The SDH Mr 65,000 flavoprotein subunit was labeled with [35S]diazobenzenesulfonate in isolated membranes but not in protoplasts. The flavoprotein subunit is thus located on the cytoplasmic side of the membrane. The location of the SDH Mr 28,000 iron-protein subunit was not definitely established, but most probably the iron-protein subunit also is located on the cytoplasmic side of the membrane. Antibodies were not obtained to the hydrophobic cytochrome b558. The cytochrome was strongly labeled with [35S]diazobenzenesulfonate in protoplasts, and labeling was also obtained with isolated membranes. Cytochrome b558 is thus exposed on the outside of the membrane. In B. subtilis SDH binds specifically to cytochrome b558, which suggests that the cytochrome is exposed also on the cytoplasmic side of the membrane. The results obtained suggest that the B. subtilis SDH is exclusively located on the cytoplasmic side of the membrane where it is bound to cytochrome b558, which spans the membrane.     

Surface-enhanced resonance Raman spectroscopy of porphyrin and metalloporphyrin species in systems with Ag nanoparticles and their assemblies

The advantages of systems with Ag nanoparticles and their assemblies for surface-enhanced resonance Raman scattering (SERRS) spectral investigation, detection and determination of porphyrin species are demonstrated. SERRS spectral detection limits of the testing porphyrin species (including porphyrin aggregates) in these systems are shown to be, on average, 10(2)-10(3) lower than detection limits by resonance Raman scattering (RRS). Systems with Ag nanoparticles modified by anionic organosulfur spacers enable us to obtain SERRS spectra of unperturbed cationic porphyrin species. In the case of thiopheneacetate-modified Ag particles prepared by laser ablation, no negative effect of the spacer on the spectral detection limit of the porphyrin was observed. Systems with isolated Ag nanoparticles allow for obtaining SERRS spectra of porphyrin species upon excitation into the Soret electronic absorption band which leads to at least a 10-fold decrease in the detection limit.     

SERRS study of the DNA binding by Ru(II) tris-(Bipyridyl) complexes bearing one carboxylic group

Surface enhanced resonance Raman scattering (SERRS) is shown to be a satisfying method to study the interaction between DNA and ruthenium complexes [Ru(bpy)(2)(Hcmbpy)][PF(6)](2), where Hcmbpy = 4-carboxy-4'-methyl-2,2'-bipyridine. Such metallic complexes are known for their fluorescence properties. To validate this spectroscopic approach we have checked that i) at a given lambda(ex), silver colloidal SERRS spectra of Ru complexes closely resemble resonance Raman spectra in aqueous solutions, intensity excepted, and ii) the DNA fragments are not altered when they are adsorbed on the Ag nanoparticles surface. This investigation shows that the intensity of the Ru complexes SERRS spectra is reduced in the presence of DNA, in particular for the specific bands assigned to the Hcmbpy ligand. This collapse demonstrates that the Ru complexes bind DNA through the Hcmbpy moiety, and intercalation is suggested as the binding mode. The DNA binding by the enantiopure Ru complexes (Delta or Lambda) is more efficient than by the racemic complexes.     

Surface-enhanced resonance Raman spectroscopy of phycocyanin and allophycocyanin

High quality surface-enhanced resonance Raman (SERR) spectra were recorded from native and denatured phycocyanin and allophycocyanin on ascorbic acid treated silver hydrosols. The visible-excited SERR and resonance Raman (RR) spectra of the phycobiliproteins were very similar, indicating a predominantly electromagnetic surface enhancement mechanism. Investigation of pH-induced denaturation ofx allophycocyanin has shown that even small differences in protein/chromophore conformational are sensitively reflected by the SERR spectra. Concerning the adsorption of the protein to the metal surface, the experiments have shown that: (i) there is limited possibility for changing protein conformation during the adsorption process, (ii) there are no changes after the protein has been adsorbed onto the silver surface and (iii) for each protein an optimal activation of the silver sol has to be found for recording proper SERR spectra. The results obtained on phycobiliproteins are also discussed in connection with the interpretation of phytochrome Raman spectra.     

Vibrational spectroscopy of excited electronic states in carotenoids in vivo. Picosecond time-resolved resonance Raman scattering.

The vibrational spectroscopy and population dynamics of excited singlet (2(1)Ag), excited triplet (3B u), and the ground (1Ag) electronic states of carotenoids in chromatophores of Chromatium vinosum (mainly spirilloxanthin and rhodopin) and of the same carotenoids in benzene solutions are examined by picosecond time-resolved resonance Raman scattering. Coherent Stokes Raman scattering from the ground states of carotenoids in chromatophores also is observed. Resonance Raman spectra of in vitro rhodopin and spirilloxanthin when compared with in vivo data demonstrate that scattering from spirilloxanthin dominates the in vivo spectrum. Comparisons of the time-dependent intensities of 2(1)Ag and 1Ag resonance Raman bands from both in vitro and in vivo carotenoids suggest that vibrationally excited levels in 1Ag are populated directly by the decay of the 2(1)Ag state and that these levels relax into a thermalized distribution in less than 50 ps. The appearance of asymmetrically broadened, ground-state resonance Raman bands supports this conclusion. Formation of the 3Bu state is observed for carotenoids in chromatophores, but not for in vitro spirilloxanthin indicating that the 3Bu state is formed by fission processes originating from the spatial organization of pigments within chromatophores. The rate at which the intensities of 2(1)Ag resonance Raman bands decay is faster for the carotenoids in vivo than for those in vitro thereby indicating that additional relaxation channels (e.g., energy transfer to bacteriochlorophylls) are present in the chromatophore. The similarity of the in vivo and in vitro 2(1)Ag resonance Raman spectra shows that no significant modifications in the vibronic coupling has been caused by the chromatophore environment.     

Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase

When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping.     

The study of microviscosity of plasma membranes of Nitella cells during rest and excitation

Changes in the microviscosity of excitable membranes was investigated using resonance Raman spectroscopy of carotenoids. The Raman resonance spectra of carotenoids in Nitella cells were excited by 514.5 nm line of an argon ion laser. The bands at 1525 cm-1, 1160 cm-1 and 1008 cm-1 were observed and they were assigned to C=C, C-C and C-CH vibrations, respectively. The rhythmic excitation of cell reduced the intensity and increased the ratios of intensity of major carotenoid bands with no noticeable shift in the position of peaks. The Arrhenius plot of relative intensity ratios of 1525 cm-1 and 1160 cm-1 bands versus reciprocal temperature showed a change of the slope in the range of 13-18 degrees C. This indicates a membrane phase transitions in which a reorientation of carotenoids species takes place. The interpretation was supported by parallel microcalorimetric and EPR measurements. The decrease of microviscosity with increasing temperature is probably caused by changes in polyene chain conformation. It is suggested that membrane microviscosity during NH4(+)-stimulated rhythmic excitation of algal cells increases, and membrane-associated carotenoids act as microviscosity-sensitive "potential sensor" for the channel.     

Ultraviolet resonance Raman spectroscopy of distamycin complexes with poly(dA)-poly(dT) and poly(dA-dT): role of H-bonding

Raman spectra are reported for distamycin, excited at 320 nm, in resonance with the first strong absorption band of the chromophore. Qualitative band assignments to pyrrole ring and amide modes are made on the basis of frequency shifts observed in D2O. When distamycin is dissolved in dimethyl sulfoxide or dimethylformamide, large (30 cm-1) upshifts are seen for the band assigned to amide I, while amides II and III shift down appreciably. Similar but smaller shifts are seen when distamycin is bound to poly(dA-dT) and poly(dA)-poly(dT). Examination of literature data for N-methylacetamide in various solvents shows that the amide I frequencies correlate well with solvent acceptor number but poorly with solvent donor number. This behavior implies that acceptor interactions with the C = O group are more important than donor interactions with the N-H group in polarizing the amide bond and stabilizing the zwitterionic resonance form. The resonance Raman spectra therefore imply that the distamycin C = O groups, despite being exposed to solvent, are less strongly H-bonded in the polynucleotide complexes than in aqueous distamycin, perhaps because of orienting influences of the nearby backbone phosphate groups. In this respect, the poly(dA-dT) and poly(dA)-poly(dT) complexes are the same, showing the same RR frequencies. Resonance Raman spectra were also obtained at 200-nm excitation, where modes of the DNA residues are enhanced. The spectra were essentially the same with and without distamycin, except for a perceptable narrowing of the adenine modes of poly(dA-dT), suggesting a reduction in conformational flexibility of the polymer upon drug binding.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes.

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000-1130 cm-1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the I portion of the membrane spectrum with a strong 1658 cm-1 band characteristic of C=C stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm-1 attributable to membrane-associated carotenoids.     

Surface-enhanced resonance Raman scattering of porphyrins on gold nanoparticles attached to silanized glass plates

Surface-enhanced resonance Raman scattering (SERRS) spectra of cationic 5,10,15,20-tetrakis(1-methyl-4-pyridyl) porphyrin (TMPyP) and anionic 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrin (TSPP) were measured from gold surfaces prepared by attaching citrate-reduced colloidal nanoparticles to glass slides silanized by 3-aminopropyltrimethoxysilane. SERRS spectra of both porphyrins obtained in a large concentration range (1 x 10(-4) to 1 x 10(-7)M) of primary solution do not show any sign of porphyrin metalation or perturbation of its native structure. Optimal adsorption time (15-20 min) and covering concentration limit (lower than 1 x 10(-5)M) of porphyrins have been estimated from the concentration and soaking time dependences of SERRS spectra.     

Chromophore of Bacteriorhodopsin is Closer to the Cytoplasmic Surface of Purple Membrane: Fluorescence Energy Transfer on Oriented Membrane Sheets

Transmembrane location of the retinal chromophore, either native or reduced in situ to a fluorescent derivative, of the purple membrane of Halobacterium halobium was investigated with fluorescence energy transfer techniques. Single sheets of purple membrane, either native or reduced with borohydride, were adsorbed on polylysine-coated glass; the orientation, whether the exposed surfaces were cytoplasmic or extracellular, was controlled by adjusting the pH of the membrane suspension before the adsorption. On the exposed surface of the reduced membrane, a layer of cytochrome c, hemoglobin, or ferritin was deposited. The rate of excitation energy transfer from the fluorescent chromophore in the membrane to the colored protein was greater when the protein was on the cytoplasmic surface of the membrane than when it was on the extracellular surface. Analysis in which uniform distribution of the protein on the surface was assumed showed that the reduced chromophore is situated at a depth of <1.5 nm from the cytoplasmic surface. The location of the native retinal chromophore was examined by depositing a small amount of tris(2,2′-bipyridyl)ruthenium(II) complex on the native membrane adsorbed on the glass. Energy transfer from the luminescent complex to the retinal chromosphore was more efficient on the cytoplasmic surface than on the extracellular surface, suggesting that the native chromophore is also on the cytoplasmic side. From these and previous results we conclude that the chromophore, whether native or reduced, of bacteriorhodopsin is located at a depth of 1.0 ± 0.3 nm from the cytoplasmic surface of purple membrane.     

Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mutations at tyrosine M210

Resonance Raman (RR) spectroscopy and low-temperature absorption spectroscopy have been used to investigate the structural changes in the reaction centers (RCs) of Rhodobacter sphaeroides induced by site-specific mutations on the tyrosine (Y) M210 residue. RCs in which Y M210 has been genetically replaced with phenylalanine (F) or leucine (L) exhibit a 5-fold decrease in their primary electron-transfer kinetics (Finkele et al., 1990). The general similarity of RR spectra of the wild-type RCs as compared to those of the two mutant RCs indicates that no significant global structural changes occur upon mutation at the level of any of the six bacteriochlorin pigments. In the RR spectra of the two mutant RCs there is a conspicuous absence of contributions from the BPheM prosthetic group, which is interpreted in terms of a change in the resonance enhancement conditions of this chromophore. Low-temperature adsorption spectroscopy reveals marked shifts in the Qx absorption band of BPheM. This shift is interpreted as arising from a destabilization of the protein in the vicinity of BPheM and accounts for the change in resonance condition for this chromophore in its RR contributions. As well, there is a 3-nm red shift of the Qy absorption band of the BChls from 803 to 806 nm for the mutant RCs. Difference RR spectra yielding structural information concerning, selectively, the primary donor (P) indicate that the structure of the P binding pocket is conserved for these mutant RCs. The tyrosine M210 is not observed to be engaged in a hydrogen bond with either of the acetyl or keto carbonyls of P.