Raman studies of conformational changes in model membrane systems

Laser Raman spectra of concentrated samples of phosphatidyl choline and phosphatidyl ethanolamine were taken at approximately 10° intervals over a temperature range of 90°–19°C. The spectral region from 30 to 3300 cm^?1 was investigated. Several new spectral features were discovered which are correlated to phospholipid liquid crystalline structure. It is shown that 1) frequency shifts occur in the $\text{PO}{}_2{}^{\mathrm{?}}$ symmetric stretch band which suggest a change in exposure of the PO₂ group to the solvent upon melting, 2) the frequency of the translational hydrocarbon mode around 150 cm^?1 appears to indicate the degree to which the hydrocarbon chain is extended, 3) the methyl and methylene stretch bands at 2890 and 2850 cm^?1 very clearly demonstrate hydrocarbon chain melting, and 4) the 720 cm^?1 band, previously assigned to the symmetric OPO diester stretch, appears to be due instead to the symmetric CN stretch of choline.     

Evidence for a krill‐rich diet from non‐destructive analyses of penguin bone

Diet strongly influences the chemistry of vertebrate soft and hard tissues. Bird bone and eggshell mineral preserve reliable records of prey consumption, even beyond the life of the predator, and analyses of hard tissues have usefully reconstructed avian diet. Here, we assess the feasibility of a non‐destructive method for distinguishing krill‐poor from krill‐rich diets in penguins. Krill (Euphausiaceae) are fluoride‐rich, and penguins that consume krill produce fluoride‐rich bones. The chemistry of bone mineral may be elucidated using Raman spectroscopy without recourse to specialised sample preparation. Published data from the diet of six penguin species were compared to a fluoride‐informative spectral band (phosphate symmetric stretch, ν₁‐PO₄^3?) in the Raman spectra of penguin humeri. Penguins that consume abundant krill (e.g. Adélie and emperor) have ν₁‐PO₄^3?‐band positions higher than 963 cm^?1, whereas penguins that primarily eat teleost fish or cephalopods (e.g. Fiordland crested, Humboldt, little blue and yellow‐eyed) have ν₁‐PO₄^3?‐band positions lower than 963 cm^?1. A krill‐rich diet can therefore be determined from the Raman spectra of penguin bones. Raman spectroscopy could be a useful supplement to existing diet analysis techniques.     

Laser Raman identification of an interaction site on DNA for arginine containing histones in chromatin.

Comparisons of the Raman spectra of DNA, chromatin, and complexes of DNA with poly-L-arginine and N-α-acetylarginine have been made. Both in native chromatin and in complexes of DNA with the arginine derivatives there is a marked decreased in the Raman intensity of the 1490±2 cm^?1 band due to guanine. Considerable evidence is presented to show that a decrease in the intensity of the 1490 cm^?1 Raman band of quanine in DNA is strong indication of a hydrogen bond being attached to the N-7 position of quanine. A specific model is presented for the interaction of the arginine residues with the guanine residues in the major groove of DNA. The Raman frequency of the histone Amide 1 band indicates that these protein molecules have a high α-helical content while the phosphate diester stretch frequency of the DNA shows the DNA to be in the B-family.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.     

A quantitative bioapatite/collagen calibration method using Raman spectroscopy of bone

Numerous calibration models were developed and tested for the quantitative analysis of collagen and bioapatite in bone using Raman spectroscopy. The ν₁ phosphate vibration at 960 cm^–1 was used as indicator of the content of bioapatite while for collagen three markers were used: the C–H₂ band at 2940 cm^–1, the amide I band at 1667 cm^–1 and the vibrations of proline and hydroxyproline at 855 and 878 cm^–1, respectively. Also a calibration model based on the PLS algorithm was developed, too. Validation of the derived calibration models indicated that the model that makes use of the height ratio of the peaks 960/(855+878) exhibits the best accuracy. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Infrared and raman studies of the methyl cis undecenoates and of the methyl undecynoates

The infrared spectra (of CCl₄ solutions) and the Raman spectra (of the neat liquids) of the eight isomeric methyl cis-undecenoates, of methyl undec-10-enoate, and of the nine isomeric methyl undecynoates have been obtained. Conjugation of the double bond with the carbonyl group lowers the wavenumber value of ν(CC) by 8 cm^?1 from the mean value of 1657 cm^?1. Conjugation of the triple bond with the carbonyl group gives rise to a single Raman band due to ν(CC) at 2241 cm^?1 whereas the non-conjugated, non-terminal triple bond gives a Fermi resonance doublet at 2234 and 2293 cm^?1.     

Raman spectroscopic study of the valinomycin--KSCN complex

This paper reports the first Raman spectroscopic study of the potassium complex of the cation-specific antibiotic valinomycin. Complete Raman spectra (140 to 3600 cm^?1) of crystalline valinomycin-KSCN and its CCl₄, CHCl₃ and C₂H₅OH solutions are presented and used to probe the structure of the complex in these environments. In all cases a single, narrow peak is observed in the ester CO stretch region (1750 to 1775 cm^?1) which contrasts strongly with the broad bands observed in solutions of uncomplexed valinomycin. This is consistent with the presence of a single conformation in which all six ester CO groups co-ordinate an enclosed potassium ion. We find that although the ester CO stretch frequencies of the complex are similar in the solid state and in non-polar solution (~1770 cm^?1) they are considerably different in the presence of polar solvents (~1756 cm^?1); this may indicate that the complexed potassium ion is still free to interact with nearby solvent ions (and possibly its counterion) through gaps in the hydrophobic “shield” provided by the hydrocarbon residues of valinomycin. In contrast the amide CO frequencies of the complex (~1650 cm^?1) are solvent-independent. These groups are apparently strongly hydrogen-bonded to provide a rather rigid, compact framework for the complex conformation.     

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy

We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5–300 mg ml^?1). No changes in the amide‐I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm^?1 and the ratios of the intensities of the Tyr bands at 856 and 837 cm^?1, the Trp bands at 870 and 877 cm^?1, and the bands at 2940 (CH stretching) and 3420 cm^?1 (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high‐concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 237–246, 2015.     

A modification for the calculation of water depth profiles in oil‐treated skin by in vivo confocal Raman microscopy

In this study, an extended calculation method for the determination of the water profiles in oil‐treated skin is proposed, which is based on the calculation of the ratio between the Raman band intensities of water (3350‐3550 cm^?1) and keratin Amide I at 1650 cm^?1. The proposed method is compared with the conventional method based on the ratio of the Raman band intensities of water (3350‐3550 cm^?1) and keratin at 2930 cm^?1. The conventional method creates artifacts in the depth profiles of the water concentration in oil‐treated skin, showing a lower amount of water in the upper and intermediate layers of the stratum corneum, which is due to the superposition of oil‐ and keratin‐related Raman bands at 2930 cm^?1. The proposed extended method shows no artifacts and has the potential to determine the water depth profiles after topical application of formulations on the skin.     

The conformation of polycytidylic acid, polyguanylic acid, polyinosinic acid, and their helical complexes in aqueous solution from laser Raman scattering

The Raman spectra of the double helical complexes of poly C–poly G and poly I–poly C at neutral p^H are presented and compared with the spectra of the constituent homopolymers. When a completely double-helical structure is formed in solution a strong sharp band at 810–814 cm^?1 appears which has previously been shown to be due to the A-type conformation of the sugar–phosphate backbone chain. By taking the ratio of the intensity of the 810–814 cm^?1 band to the intensity of the 1090–1100 cm^?1 phosphate vibration, one can obtain an estimate of the fraction of the backbone chain in the A-type conformation for both double-stranded helices and self-stacked single chains. This type of information can apparently only be obtained by Raman spectroscopy. In addition, other significant changes in Raman intensities and frequencies have been observed and tabulated: (1) the Raman intensity of certain of the ring vibrations of guanine and hypoxanthine bases decrease as these bases become increasingly stacked (Raman hypochromism), (2) the Raman band at 1464 cm^?1 in poly I is asigned to the amide II band of the cis-amide group of the hypoxanthine base. It shifts in frequency upon base pairing to 1484 cm^?1, thus permitting the determination of the fraction of I–C pairs formed.     

Structural investigation of poly d(BrU-A) by ultraviolet resonance raman spectroscopy

The resonance Raman spectra of a DNA containing bromodeoxy-uridine (BrdUrd), the poly d(BrU-A), are reported, using U.V. laser as a source of excitation. The conformational change from the ordered, base paired form of poly d(BrU-A) (at 25°C) to the melted form at high temperature (63°C) is reflected in a pronounced hyperchromism of Raman bands at 1627 cm^?1, 1352 cm^?1 and 1230 cm^?1. Particularly the band at 1627 cm^?1 assigned to the vibrations of C4 carbonyl which is hydrogen bonded to adenine increases strongly its intensity upon melting. This represents a new approach for a detection of base unpairing and of modifications in geometry of selective molecules (BrdUrd) in a DNA chain in dilute solutions (10^?4 M).     

Conformational geometry and vibrational frequencies of nucleic acid chains.

Normal coordinate analysis of diethyl phosphate has been made, which predicts all observed Raman frequencies in the range 170–1300 cm^?1. The force constants from this calculation have been transferred to a vibrational calculation for a simplified model of the backbone of nucleic acids, which also involves the ? O? PO₂^?? O phosphate group and the ? C₅′? C₄′? C₃′? linkage of the ribose. The coordinates of these atoms are those recently given by Arnott and Hukins, which place the ribose ring of B-DNA in a C₃′-exo conformation. This simple polymer model appears to be able to describe adequately the frequency-dependent changes observed in the Raman spectra arising from the backbone vibrations of nucleic acid in going from the B- to A-form. The symmetric ? O? P? O? diester stretch increases in frequency from about 787 cm^?1 in the B-form to 807 cm^?1 in the A-form. The increased frequency characteristic of the A-form is due to the combining of the diester stretch with vibrations involving the C₅′, C₄′, and C₃′ nuclei. The frequency of the symmetric ? O? P? O? diester stretch is shown to be very dependent on the conformation of the ribose ring, indicating that in polynucleotides the ribose ring takes on one of two rigid conformations: C₃′-endo for A-form or C₃′-exo for B-form and “disordered” polynucleotides. The calculation lends confirmation to the atomic coordinates of Arnott and Hukins since the use of other geometries with the same force constants failed to give results in agreement with experimental evidence. The calculations also demonstrate the lowering effect of hydration on the anionic PO stretching frequencies. Experimental results show that the 814-cm^?1 band observed in the spectra of 5′GMP gel arises from a different vibrational mode than that of the 814-cm^?1 band of A-DNA.     

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 same technique. The fluidity of the membrane also manifests itself in the amide 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.     

Raman intensities of carbon-carbon stretching modes in a model membrane

Laser-Raman spectra of L-α-dimyristoylphosphatidylcholine (DMPC) liposomes in the spectral range 1000–1200 cm^?1 were obtained as a function of temperature from ?80 to +50°C. The triplet found in this spectral region was resolved into Lorentzian components by means of an iterative computer program. The peak intensities, band widths, and band areas of the resolved 1062 cm^?1 and 1130 cm^?1 bands, assigned to CC stretching vibrations of trans segments, were evaluated as a function of temperature. While the peak intensities of the bands decrease substantially with temperature, the band widths show a considerable increase. The change in band areas is therefore smaller than the change in peak heights. Experiments with all trans carboxylic acids showed that in these compounds the area of the Raman bands at 1062 cm^?1 and 1130 cm^?1 is proportional to the number of trans bonds. The variation with temperature of the number of trans and gauche bonds in the studied phospholipid is reflected by the change of the area of the 1130 cm^?1 Raman band.     

Electrostatically induced change of the conformational order of charged lipid membranes

Raman spectroscopy and X-ray diffraction are used to investigate the influence of surface charges on the structure of ionizable lipid membranes of dimyristoylmethylphosphatidic acid. The membrane surface charge density is regulated by varying the pH of the aqueous phase. Changes of the conformational order of the lipid chains are determined from the intensity of the CC stretch chain vibrations around 1100 cm^?1 in a lipid Raman spectrum. In going from an electrical neutral to a negatively charged membrane, the conformational order is reduced by 5% in the ordered and by 9% in the fluid membrane phase, corresponding to 0.6 and 0.8 CC bonds, respectively, which change from a trans to a gauche conformation. The electrostatically induced conformational change is mainly concentrated at the lipid chain ends as indicated by the spectral variations of the 890 cm^?1 CH₃ rocking band of the chain termini. The X-ray diffraction experiments show that increasing the surface charge density in the ordered membrane phase leads to a lateral expansion of the packing of the lipid polar groups, whereas the packing of the lipid chains in a plane perpendicular to the chain axes remains constant, indicating an increase of the tilt of the lipid chains from δ = 10° (pH 3) to δ = 27° (pH 9).     

DNA Methylation Detection Using Resonance and Nanobowtie-Antenna-Enhanced Raman Spectroscopy

We show that DNA carrying 5-methylcytosine modifications or methylated DNA (m-DNA) can be distinguished from DNA with unmodified cytosine by Raman spectroscopy enhanced by both a bowtie nanoantenna and excitation resonance. In particular, m-DNA can be identified by a peak near 1000 cm^?1 and changes in the Raman peaks in the 1200–1700 cm^?1 band that are enhanced by the ring-absorption resonance. The identification is robust to the use of resonance Raman and nanoantenna excitation used to obtain significant signal improvement. The primary differences are three additional Raman peaks with methylation at 1014, 1239, and 1639 cm^?1 and spectral intensity inversion at 1324 (C₅=C₆) and 1473 cm^?1 (C₄=N₃) in m-DNA compared to that of DNA with unmodified cytosine. We attribute this to the proximity of the methyl group to the antenna, which brings the (C₅=C₆) mode closer to experiencing a stronger near-field enhancement. We also show distinct Raman spectral features attributed to the transition of DNA from a hydrated state, when dissolved, to a dried/denatured state. We observe a general broadening of the larger lines and a transfer of spectral weight from the ～1470 cm^?1 vibration to the two higher-energy lines of the dried m-DNA solution. We attribute the new spectral characteristics to DNA softening under high salt conditions and find that the m-DNA is still distinguishable via the ～1000 cm^?1 peak and distribution of the signal in the 1200–1700 cm^?1 band. The nanoantenna gain exceeds 20,000, whereas the real signal ratio is much less because of a low average enhanced region occupancy even with these relatively high DNA concentrations. It is improved when fixed DNA in a salt crystal lies near the nanoantenna. The Raman resonance gain profile is consistent with A-term expectations, and the resonance is found at ～259 nm excitation wavelength.     

Raman scattering of collagen, gelatin, and elastin.

The Raman spectra of collagen, gelatin, and elastin are presented. The Raman lines in the latter two spectra are assigned by deuterating the amide N-H groups in gelatin and by studying the superposition spectra of the constituent amino acids. Two lines appear at 1271 and 1248 cm^?1 in the spectra of collagen and gelatin that can be assigned to the amide III mode. Possibly, the appearance of two amide III lines is related to the biphasic nature of the tropocollagen molecule, i.e., proline-rich (nonpolar) and proline-poor (polar) regions distributed along the chain. The melting, or collagen-to-gelatin transition, in water-soluble calf skin collagen is studied and the 1248-cm^?1 amide III line is assigned to the 3₁ helical regions of the tropocollagen molecule. Elastin is thought to be mostly random and the Raman spectrum confirms this assertion. Strong amide I and III lines appear at 1668 and 1254 cm^?1, respectively, and only weak scattering is observed at 938 cm^?1. These features have been shown to be characteristic of the disordered conformation in proteins.     

Resonance coherent anti-Stokes Raman scattering (CARS) spectra of flavin adenine dinucleotide,riboflavin binding protein and glucose oxidase

Coherent anti-Stokes Raman scattering spectra, in resonance with the isoalloxazine visible electronic transition, have been obtained down to 300 cm^?1 for flavin adenine dinucleotide, riboflavin binding protein and glucose oxidase, in H₂O and D₂O. Several isoalloxazine vibrational modes can be identified by analogy with those of uracil. Of particular interest is a band at ～1255 cm^?1 in H₂O, which is replaced by another at ～1295 cm^?1, in D₂O. The H₂O band appears to be a sensitive monitor of H-bonding of the N3 isoalloxazine proton to a protein acceptor group. It shifts down by 10 cm^?1 in riboflavin binding protein, and disappears altogether in glucose oxidase. Other band shifts, of 3–5 cm^?1, are similar for the two flavoproteins, and may reflect environmental changes between aqueous solution and the protein binding pockets.     

A vibrational spectroscopic study of the self-association of 5′-GMP in aqueous solution

The Raman spectra of highly concentrated solutions of 5′-GMP at neutral and acid pH were recorded in order to better characterize the structure of the self-aggregates formed in these solutions and their melting behavior. Vibrational coupling of the C?O stretching vibrations in tetrameric units at neutral pH is shown to yield a characteristic pattern of two Raman bands at ca. 1730 and 1680 cm^?1 (1708 and 1664 cm^?1 in D₂O), and an iractive mode at 1678 cm^?1 in D₂O. From the intensity of the 1730-cm^?1 band, proportional to tetramer concentration, and that at 1485 cm^?1, which reflects the stacking of the bases, the thermal stability of the self-associates formed at neutral pH is shown to be higher for stacked tetramers. At acid pH, the melting of the helical aggregates responsible for the formation of a gel is preceded by the freeing of the hydrogen-bonded phosphate groups, accompanied by a change of conformation from C3′-endo to C2′-endo in some of the associated ribose units. Previous spectroscopic results suggesting the formation of tetramers as an intermediate step in the melting of the gel were not reproduced in this study.     

Resonance Raman on the purple intermediates of the flavoenzyme D-amino acid oxidase

Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm^?1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm^?1 in the RR spectrum with D-alanine was shifted to 1675 cm^?1 upon [¹⁵N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm^?1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.