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.     

Interaction of Calf-Thymus DNA With Trivalent La,Eu, and Tb Ions. Metal Ion Binding,DNA Condensation and Structural Features

Abstract

The interaction of calf-thymus DNA with La³⁺, Eu³⁺ and Tb³⁺ has been investigated in aqueous solution at pH 6.5, using metal/DNA(P) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4 and 1/2. Correlations between FTIR spectral changes and DNA structural properties have been established. At low metal/DNA(P) (r) 1/80, the metal ions bind mainly to the PO^? ₂ groups of the backbone, resulting in increased base-stacking interaction and duplex stability. At (r) 1/40 and 1/20, metal ion binding to the PO^? ₂ and the guanine N-7 site (chelation) predominates with minor perturbations of the A-T base pairs. Evidence for this comes from the displacement of the band at 1712 cm^?1 (T,G) towards a lower frequency and the PO^? ₂ antisymmetric band at 1222 cm^?1 towards a higher frequency. At higher metal/DNA(P) ratio, r> 1/20, DNA begins to condensate and drastic structural changes occur, which are accompanied by the shift and intensity changes of several G-C and A-T absorption bands. No major departure from B-DNA conformation was observed before and after DNA condensation eventhough some local structural modifications were observed. A comparison with the Cu-DNA complexes (denaturated DNA) shows some degree of helical destabilizition of the biopolymer in the presence of lanthanide ions.     

Resonance raman spectroscopy of iron (3)--ovotransferrin and iron (3)--human serum transferrin

We report the resonance Raman spectra in the frequency range 300–1800 cm^?1 of Fe (III)-ovotransferrin and Fe (III)-human serum transferrin in aqueous solution at about 10^?4M protein concentration. This is the first observation of resonance Raman scattering ascribable to amino acid ligand vibrational modes of a nonheme iron protein. The resonance Raman spectra of the transferrins are similar except that the resonance band near 1270 cm^?1 is shifted to a higher frequency for Fe(III)-human serum transferrin than that for Fe(III)-ovotransferrin. The resonance Raman bands observed near 1170, 1270, 1500 and 1600 cm^?1 may reflect resonance enhancement of p-hydroxy-phenyl frequencies of tyrosine residues and/or imidazolium frequencies of histidine residues.     

The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.     

Conformational dependence of the Raman scattering intensities from polynucleotides. 3. Order-disorder changes in helical structures

Raman spectra are presented on ordered and presumably helical structures of DNA and RNA as well as the poly A·poly U helical complex, polydAT, and the helical aggregates of 5′-GMP and 3′-GMP. The changes in the frequency and the intensity of the Raman bands as these structures undergo order-disorder transitions have been measured. In general the changes we have found can be placed into three categories: (1) A reduction in the intensities of certain ring vibrations of the polynucleotide bases is observed when stacking or ordering occurs (Raman hypochromism). Since the ring vibrational frequencies are different for each type of base, we have been able to obtain some estimate of average amount of order of each type of base in partially ordered helical systems. (2) A very large increase in the intensity of a sharp, strongly polarized band at about 815 cm^?1 is observed when polyriboA and polyriboU are formed into a helical complex. Although this band is not present in the separated chains at high temperature, a broad diffuse band at about 800 cm^?1 is present. The 815 cm^?1 band undoubtedly arises from the vibrations of the phosphate-sugar portions of the molecule and provides a sensitive handle to the back-bone conformation of the polymer. This band also appears upon ordering of RNA, formation of the helical aggregate of 5′-riboGMP, and to some extent in the selfstacking of the polyribonucleotides polyA, polyU in the presence of Mg⁺⁺, PolyC, and polyG. No such intense, polarized band is found, however, in ordered DNA, polydAT, or the 3′-riboGMP aggregate, although there is a conformationally independent band at about 795 cm^?1 in DNA and polydAT. (3) Numerous frequency changes occur during Conformational changes. In particular the 1600–1700 cm^?1 region in D₂O shows significant conformationally dependent changes in the C?O stretching region analogous to the changes in this region which have been observed in these substances in the infrared. Thus, Raman scattering appears to provide a technique for simultaneously observing the effects of base stacking, backbone conformation and carbonyl hydrogen bonding in nucleic acids in moderately dilute (10–25 mg/ml) aqueous solutions.     

Contribution of the Resonance Raman Spectroscopy to the Identification of Z DNA

Abstract

Poly(dG-dC)?poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition.

With 257 nm excitation wavelength the 1580 cm^?1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm^?1 and at 1193 cm^?1 and the bands of the cytosines at 780 cm^?1, 1242 cm^?1 and 1268 cm^?1.

By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Raman Spectroscopy Study of the B-Z Transition in (dG-dC)n · (dG-dC)n and a DNA Restriction Fragment

Abstract

The B to Z conformational transition of (dG-dC)_n·(dG-dC)_n and a 157 bp DNA restriction fragment were followed using Raman spectroscopy. The 157 bp DNA has a 95 bp segment from the E. coli lactose operon sandwiched between 26 and 32 bp of (dC-dG) sequences. Raman spectra of the DNAs were obtained at varying sodium chloride concentrations through the region of the transition. A data analysis procedure was developed to subtract the background curves and quantify Raman vibrational bands. Profiles of relative intensity vs. sodium chloride concentration are shown for bands at 626, 682, 831–833 and 1093 cm^?1. Both (dG-dC)_n·(dG-dC)_n and the 157 bp DNA show changes in the guanine vibration at 682 cm^?1 and backbone band at 831–3 cm^?1 preceeding a highly cooperative change in the 1093 cm^?1 PO 7 vibration. This result indicates that there are at least two conformational steps in the B to Z conformational pathway.

We review the effect of the (dC-dG) portion of the 157 bp DNA on the 95 bp segment. Comparison of Raman spectra of the 157 bp DNA, the 95 bp fragment and (dG-dC)_n·(dG- dC)_n indicate that in 4.5 M NaC/the (dC-dG) segments are in a Z-conformation. Base stacking in the 95 bp portion of the 157 bp DNA appears to maintain a B-type conformation. However, a substantial portion of this region no longer has a B-type backbone vibration.     

Intensity Changes of Base and Backbone Raman Modes in the B to Z Transition of poly(dG-dm5C)

Abstract

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm-⁵C). The transition midpoint was about 37°C for a solvent containing 20 mM Mg²⁺. A 10-fold change in Mg²⁺ concentration altered the transition midpoint by at least 60°C. Raman spectra of the B and Z forms of poly(dG-dm⁵C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm^?1 guanine mode and 835 cm^?1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm^?1, 805 and 819 cm¹. Several bands unique to poly(dG-dm⁵C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

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).     

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.     

Study of furanose ring flexibility in polynucleotide chains using Raman spectra analysis

The bands within the range of 800–850 cm^?1 of Raman spectra of polynucleotides sensitive to the change in conformation of sugar–phosphate backbone are analyzed theoretically. The bands are interpreted as the appearance of a quasi-local deoxyribose vibrational mode whose frequency is dependent on the ring puckering. The localization region of the vibrational mode is pointed out. The theory establishes a relationship between the observed spectral intensity and the population of deoxyribose conformational states described in the framework of the pseudorotation concept. The approach developed allowed one to describe the band shapes and their temperature behavior, and to determine the pseudorotation potential of deoxyribose in the helix B -form of A · T containing polynucleotides. Using the analysis of Raman spectra of DNA fibers in water–ethanol mixture the deoxyribose flexibility during the B -A transition is investigated in terms of the population of conformers and effective potential. It is shown that N- and S-type deoxyribose conformers are populated in the DNA B -form (those of the S-type are preferable), whereas N-type conformers are primarily populated in the DNA A -form.     

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.     

Low-frequency Raman spectra of lysozyme.

New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm^?1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm^?1. However, a low-frequency band at 25 cm^?1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.     

An FT-IR Study of DNA and RNA Conformational Transitions at Low Temperatures

Abstract

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO₂ and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant.

Marker infrared bands for the B conformer have been found to be the strong band at 825 cm^?1 (sugar conformer mode) and a band with medium intensity at 690 cm^?1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm^?1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm^?1 and at 665-600 cm^?1.     

Studies on determining conformational order in n-alkanes and phospholipids from the 1130 cm−1 raman band

Conformational disorder in lipid bilayer systems is commonly measured with reference to the intensity of the 1130 cm^?1 Raman band. However, estimates of the concentration of gauche bonds may vary by a factor of six according to the model used to relate intensity and concentration. In an effort to narrow the wide range in these estimates, we have measured the intensity of the 1130 cm^?1 band of crystalline n-C₂₁H₄₄ in its orthorhombic and hexagonal phases. On transition to the hexagonal phase, the intensity of the 1130 cm^?1 band is much reduced. It is assumed that the observed intensity reduction results from the introduction of gauche bonds whose number can be independently estimated from other features in the Raman and infrared spectra. From these measurements we conclude that the intensity of the 1130 cm^?1 band is not linearly related to the concentration of gauche bonds and that a disproportionately large decrease in the 1130 cm^?1 band intensity results from the introduction of a low concentration of gauche bonds. Thus previous estimates of gauche bond concentrations based on the assumption of a linear relation have tended to greatly overestimate the gauche bond concentration. These results derived from experiment are in accord with those of Pink et al. (Pink, D.A., Green, T.J. and Chapman, D. (1980) Biochemistry 19, 349–356) derived from theory.     

The Raman spectra of Bence-Jones proteins. Disulfide stretching frequencies and dependence of Raman intensity of tryptophan residues on their environments

The Raman spectra of Bence-Jones proteins (BJP) were measured for their native and denatured states. All of the native BJPs investigated gave amide I at 1670–1675 cm^?1 and amide III at 1242–1246 cm^?1. Although the amide I was shifted to 1667 cm^?1 upon the LiBr, acid, and thermal denaturation, as expected, the amide III frequency was unaltered, indicating that the antiparallel β- and disordered structures of BJP provide amide III at almost the same frequencies. The intensity of the 880-cm^?1 line of native BJP was relatively intense compared with that of amino acid mixed solution in which the mole ratios of Trp, Phe, and Tyr were adjusted to reproduce the corresponding ratios of BJP. However, the intensity was evidently reduced upon LiBr, acid, and thermal denaturation, approaching that of the amino acid mixture. Thus, the intensity of the 880-cm^?1 line is proposed as a practical probe for the environment of Trp residues. The pH dependence of the intensity of the 880-cm^?1 line suggests that one of two buried Trp residues is exposed between pH 4 and 3.2 and the other between pH 3.2 and 1.4. The variable fragment (V_L) of BJP (Tod) exhibited a S? S stretching Raman line at 525 cm^?1. Provided that the crystallographic data of the V_L of BJP is applicable to V_L of BJP (Tod), the 525 cm^?1 of the S? S stretching frequency should be assigned to a TGG conformation of linkage, but not to the AGT or AGG conformation. This supports Sugeta's model rather than Scheraga's model.     

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.     

Changes in Raman vibrational bands of calf thymus DNA during the B-to-A transition

The B -to-A conformational transition of calf thymus DNA fibers was followed employing Raman spectroscopy. The transition was induced by soaking DNA fibers in water/ethanol mixtures increasing from 60 to 85% ethanol (v/v). Intensity changes of 17 Raman vibrational bands were quantified in the region from 400 to 860 cm^?1. Two bands at 500 and 784 cm^?1 were employed as internal standards. These bands do not appear to change in intensity with ethanol concentration. Large intensity changes relative to these two bands are observed between 70 and 74% ethanol for backbone vibrations at 708, 808, and 835 cm^?1, and base vibrations at 682, 730, and 750 cm^?1. These results indicate that a highly cooperative conformational change takes place between different portions of DNA in the B -to-A transition. Relative intensity changes preceding the onset of the major transition are observed in only two bands; at 835 cm^?1, assigned to a ribose–phosphate vibration, and at 750 cm^?1, assigned to thymine. The implications of these pretransition changes are discussed.     

Raman studies of nucleic acids. VII. Poly A-poly U and poly G-poly C

Laser-excited Raman spectra of the double-helical complexes poly A·poly U and poly G·poly C are reported for ²H₂O and H₂O solutions. The spectra are discussed in relation to their use as quantitative reference spectra for determining the dependence of the Raman scattering of RNA on secondary structure. The Raman line at 815 cm^?1, due to the phosphodiester group, exhibits the same intrinsic intensity in spectra of poly A·poly U and poly G·poly C and is thus dependent only upon the amount of ordering of the helix and not on the kinds of nucleotides involved. The hypochromic Raman lines in spectra of poly A·poly U are identified and their intensity changes are determined quantitatively over the temperature range 32–85°C. Comparison of the spectra in the 1500–1750 cm^?1 region reveals that the Raman lines from carbonyl group vibrations of uracil are about sevenfold more intense than those of guanine and cytosine for both paired and unpaired states and will thus dominate the spectra of RNA. The Raman frequencies in this region are also compared with previously reported infrared frequencies and give evidence of being strongly perturbed by base-stacking interactions in the helices.     

Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

The first investigation of the dynamics of a redox transition of an electron-transfer enzyme by time-resolved resonance Raman spectroscopy in combination with pulse-radiolytical reduction is described by an application to cytochrome c. A long-lived transient state is observed upon reduction of the alkaline form of cytochrome c as a distinct frequency shift of one resonance Raman band. From the frequency in the stable oxidized state, 1567 cm^?1, this particular resonance Raman band shifts within less than 1 μs to 1533 cm^?1 in the transient reduced state, which has a lifetime longer than 20 ms but shorter than a few seconds. Finally, in the stable reduced state, this band is located at 1547 cm^?1. According to a previous normal coordinate analysis, this resonance Raman band can be assigned predominantly to a stretching mode of the outermost C-C bonds in the four pyrrole rings of porphyrin. This vibrational mode is influenced by the protein most directly through the covalent thioether linkages of two cysteines to porphyrin. We interpret the long lifetime of the transient state as due to the slow return of Met-80 as sixth ligand to the heme iron upon reduction of the alkaline form of cytochrome c.