Conformational dependence of the Raman scattering intensities from polynucleotides

The intensity of Raman scattering from the various Raman active vibrations of poly-(riboadenylic acid), poly(ribocytidylic acid), poly(ribouridylic acid), and poly(riboinosinic acid) in moderately dilute solutions were examined as the temperature was changed to alter their conformation. It was found that certain highly intense, highly polarized Raman bands from the totally symmetric, i.e., in-plane, ring vibrations of the nucleic acid bases become less intense as the chains become more ordered in solution. Since these vibrations occur at frequencies which are markedly different for each type of base, Raman spectroscopy appears to provide a new method for the characterizing of the average conformation of each of the bases in solution. A theory for the resonant Raman effect is given in which it is shown that, a decrease in resonant Raman intensity is to be expected if one obtains a decrease in the intensity of the corresponding ultraviolet absorption band with which the incident light is resonant. If it is assumed that certain Raman bands derive their intensity predominantly from the first few ultraviolet absorption intensities, then a qualitative explanation of our observed conformational dependence of the ordinary Raman intensities can be obtained.     

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.     

Structural characterization of cytochrome c peroxidase by resonance Raman scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.     

Calculation of small angle scattering intensities from molecular dynamics simulation

A method is presented to efficiently calculate small-angle neutron and X-ray solution scattering intensities from explicit - atom model of macromolecules and the surrounding solvent.     

Resonance-enhanced Raman scattering from the molybdenum center of xanthine oxidase

The molybdenum center of xanthine oxidase has been examined by resonance Raman spectroscopy. Making use of the long-wavelength absorption of the reduced molybdenum center in complex with violapterin (the product of enzymic action of lumazine), resonance Raman spectra were obtained using laser excitation at 676.4 nm. Several internal vibrational modes of violapterin were found to be resonance-enhanced, and a number of bands in the 250-1100 cm-1 range, presumably arising from vibrational modes of the molybdenum coordination sphere, were also observed. Upon substitution of 18O for 16O in the molybdenum coordination sphere, bands at 1469, 853, 517, 325, and 276 cm-1 exhibited shifts of 5-12 cm-1 to lower energy. By analogy to previous vibrational studies of Mo-O-Mo and Mo-O-R model compounds, the 853, 517, and 276 cm-1 frequencies were judged consistent with a labeled Mo-O-R linkage of the complexed violapterin. More importantly, the relatively small frequency shifts observed in these and other vibrations upon incorporation of 18O are very similar to those observed by others for 18O-labeled phenol and metal-phenolate complexes (Pinchas, S., Sadeh, D., and Samuel, D. (1965) J. Phys. Chem. 69, 2259-2264; Pyrz, W. J., Rue, L. A., Stern, L. J., and Que, L. J., Jr. (1985) J. Am. Chem. Soc. 107, 614-620) that model iron-tyrosinate proteins. The relatively small isotope-induced frequency shifts in multiple bands are thus interpreted as resulting from vibrational mixing of internal coordinates involving the oxygen atom with internal ring motions of the aromatic species. No oxygen isotope-sensitive bands were observed in the 900-1100 cm-1 region where Mo = O stretching modes typically occur. In agreement with the conclusions of previous workers (Davis, M.D., Olson, J. S., and Palmer, G. (1982) J. Biol. Chem. 257, 14730-14737) we interpret our results to indicate that the absorption band appearing upon complexation of violapterin with the molybdenum center of reduced xanthine oxidase is a molybdenum-to-violapterin charge-transfer band. These results, as well as several other lines of evidence, are consistent with direct coordination of violapterin to molybdenum in the charge-transfer complex via the 7-hydroxyl group (i.e. the hydroxyl group introduced into substrate by the enzyme). The Mo=O stretching mode of the complex is presumably not resonance enhanced because it is orthogonal to the charge-transfer electronic transition, suggesting that coordination of violapterin is cis to the oxo group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple‐helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ～60‐80°C for hydrated samples. We report high temperature Raman spectra for freeze‐dried cartilage samples that show an increase in laser‐excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H₂O is retained within the freeze‐dried tissue. This is confirmed by thermogravimetric analysis that show 5‐7 wt% H₂O remaining within freeze‐dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re‐hydration following recovery indicate that the capacity of the denatured collagen to re‐absorb water is reduced. Our results are important for revealing the presence of bound H₂O within the collagen component of connective tissue even after freeze‐drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.     

Excited-state structure and isomerization dynamics of the retinal chromophore in rhodopsin from resonance Raman intensities.

Resonance Raman excitation profiles have been measured for the bovine visual pigment rhodopsin using excitation wavelengths ranging from 457.9 to 647.1 nm. A complete Franck-Condon analysis of the absorption spectrum and resonance Raman excitation profiles has been performed using an excited-state, time-dependent wavepacket propagation technique. This has enabled us to determine the change in geometry upon electronic excitation of rhodopsin's 11-cis-retinal protonated Schiff base chromophore along 25 normal coordinates. Intense low-frequency Raman lines are observed at 98, 135, 249, 336, and 461 cm-1 whose intensities provide quantitative, mode-specific information about the excited-state torsional deformations that lead to isomerization. The dominant contribution to the width of the absorption band in rhodopsin results from Franck-Condon progressions in the 1,549 cm-1 ethylenic normal mode. The lack of vibronic structure in the absorption spectrum is shown to be caused by extensive progressions in low-frequency torsional modes and a large homogeneous linewidth (170 cm-1 half-width) together with thermal population of low-frequency modes and inhomogeneous site distribution effects. The resonance Raman cross-sections of rhodopsin are unusually weak because the excited-state wavepacket moves rapidly (approximately 35 fs) and permanently away from the Franck-Condon geometry along skeletal stretching and torsional coordinates.     

Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between (-) 10 and 5 degrees C (low-temperature transition), 10 and 22 degrees C (middle-temperature transition), and 32 and 40 degrees C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14 degrees C). Second, the middle-temperature transition shifts up to the range of about 20-32 degrees C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of about 15-40 degrees C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties.     

Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

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.     

Structural changes of human RNase L upon homodimerization investigated by Raman spectroscopy

RNase L, a key enzyme in the host defense system, is activated by the binding of 2'-5'-linked oligoadenylates (2-5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2-5 pA(4) and three tetramers with 3'-end AMP units replaced with ribo-, arabino- and xylo-configured phosphonate analogs of AMP (pA(3)X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α-β structural switch may fix domain positions in the hinge region (residues ca. 336-363) during homodimer formation.     

Raman scattering from nucleic acids adsorbed at a silver electrode

Adsorption of nucleic acids at a silver electrode polarized to -0.6 to -0.1 V (vs. Ag/AgCl) was investigated by means of surface enhanced Raman scattering (SERS) spectroscopy. Single-stranded polyriboadenylic acid and thermally denaturated DNA adsorbed at the silver electrode yield two intense bands at 734 and 1335 cm-1 on the SERS spectra. These bands, assigned to the vibrations of adenine residue rings, were much less intense if the SERS spectra were recorded for double-helical complex polyadenylic X polyuridylic acid and native DNA. Moreover, the courses of alkaline denaturation of DNA and its digestion by deoxyribonuclease I were observed by SERS spectroscopy. The results were interpreted as support for the view that intact double-helical segments of nucleic acids are not denatured or destabilized due to their adsorption at the positively charged and roughened surface.     

Intermolecular potentials for alpha-glycine from Raman and infrared scattering measurements

The frequencies of intermolecular modes in alpha-glycine-d0 and -d5 have been measured at 300 and 85 K by Raman and infrared scattering techniques. These frequencies were analyzed in terms of simple analytic interatomic potentials. Buckingham potentials were assumed for the nonbonded and hydrogen-bond interactions, and Coulomb and screened Coulomb potentials were assumed for the electrostatic interactions. The observed frequencies are well described by the simple model and the parameters of the hydrogen-bond potentials and the molecular charge distribution were determined from the analysis.     

Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.     

Conformational homogeneity and excited-state isomerization dynamics of the bilin chromophore in phytochrome Cph1 from resonance Raman intensities

The ground-state structure and excited-state isomerization dynamics of the P_r and P_fr forms of phytochrome Cph1 are investigated using resonance Raman intensity analysis. Electronic absorption and stimulated resonance Raman spectra of P_r and P_fr are presented; vibronic analysis of the Raman intensities and absorption spectra reveals that both conformers exist as a single, homogeneous population of molecules in the ground state. The homogeneous and inhomogeneous contributions to the overall electronic broadening are determined, and it is found that the broadening is largely homogeneous in nature, pointing to fast excited-state decay. Franck-Condon displacements derived from the Raman intensity analysis reveal the initial atomic motions in the excited state, including the highly displaced, nontotally symmetric torsional and C₁₅–H HOOP modes that appear because of symmetry-reducing distortions about the C₁₄–C₁₅ and C₁₅=C₁₆ bonds. P_fr is especially well primed for ultrafast isomerization and torsional Franck-Condon analysis predicts a <200 fs P_fr → P_r isomerization. This time is significantly faster than the observed 700 fs reaction time, indicating that the P_fr S₁ surface has a D-ring rotational barrier caused by steric interactions with the protein.     

Depolarization of Raman scattering from some nucleotides of RNA and DNA

Depolarization ratios of Raman bands, excited at 488.0 nm, of guanosine-5′-monophosphoric 4 acid, cytidine-5′-monophosphoric acid, adenosine-5′-monophosphoric acid, thymidine-5′-monophosphoric acid, and uridine-5′-monophosphoric acid have been measured in their H₂O and D₂O solutions in the spectral region from 300 to 1800 cm^?1. For comparison, the disodium salt of 2′-deoxyadenosine-5′-monophosphoric acid was also subjected to the depolarization measurement in its H₂O solution. The results have been correlated with possible orientations of the principal axes of the Raman scattering tensors as well as with the relative magnitudes of the tensor components. Results should be useful for future polarized Raman studies of synthetic and natural DNA. © 1993 John Wiley & Sons, Inc.