Low-frequency dynamics and Raman scattering of crystals, of B-, A-, and Z-DNA, and fibers of C-DNA

Normal modes of vibration of DNA in the low-frequency region (10-300 cm-1 interval) have been identified from Raman spectra of crystals of B-DNA [d(CGCAAATTTGCG)], A-DNA [r(GCG)d(CGC) and d(CCCCGGGG)], and Z-DNA [d(CGCGCG) and d(CGCGTG)]. The lowest vibrational frequencies detected in the canonical DNA structures--at 18 +/- 2 cm-1 in the B-DNA crystal, near 24 +/- 2 cm-1 in A-DNA crystals, and near 30 +/- 2 cm-1 in Z-DNA crystals--are shown to correlate well with the degree of DNA hydration in the crystal structures, as well as with the level of hydration in calf thymus DNA fibers. These findings support the assignment [H. Urabe et al. (1985) J. Chem. Phys. 82, 531-535; C. Demarco et al. (1985) Biopolymers 24, 2035-2040] of the lowest frequency Raman band of each DNA to a helix mode, which is dependent primarily upon the degree of helix hydration, rather than upon the intrahelical conformation. The present results show also that B-, A-, C-, and Z-DNA structures can be distinguished from one another on the basis of their characteristic Raman intensity profiles in the region of 40-140 cm-1, even though all structures display two rather similar and complex bands centered within the intervals of 66-72 and 90-120 cm-1. The similarity of Raman frequencies for B-, A-, C-, and Z-DNA suggests that these modes originate from concerted motions of the bases (librations), which are not strongly dependent upon helix backbone geometry or handedness. Correlation of the Raman frequencies and intensities with the DNA base compositions suggests that the complex band near 90-120 cm-1 in all double-helix structures is due to in-plane librational motions of the bases, which involve stretching of the purine-pyrimidine hydrogen bonds. This would explain the centering of the band at higher frequencies in structures containing G.C pairs (greater than 100 cm-1) than in structures containing A.T pairs (less than 100 cm-1), consistent with the strengths of G.C and A.T hydrogen bonding.     

Low frequency Raman spectra of Z-DNA

We have performed a Raman study of the low frequency modes in three oligo- and polynucleotides in Z-conformation, and we compare the spectra of these samples to those of two polynucleotides in B-conformation. In Z-DNA we find 5 intrahelical modes below 200 cm-1, in addition to the interhelical mode near 30 cm-1 which is only observed in crystalline samples. The most prominent intrahelical mode has a frequency of about 105 cm-1, close to the frequency of the strongest intrahelical mode in A-and B-DNA. The sequence dependence of the frequency of this mode is considerably larger than for the same mode in B-DNA. The other modes are less pronounced, and their frequency variations with base sequence are within the experimental accuracy.     

Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA.

Raman spectra of poly(dG-dC) . poly(dG-dC) in D2O solutions of high (4.0M NaCl) and low-salt (0.1M NaCl) exhibit differences due to different nucleotide conformations and secondary structures of Z and B-DNA. Characteristic carbonyl modes in the 1600-1700 cm-1 region also reflect differences in base pair hydrogen bonding of the respective GC complexes. Comparison with A-DNA confirms the uniqueness of C = O stretching frequencies in each of the three DNA secondary structures. Most useful for qualitative identification of B, Z and A-DNA structures are the intense Raman lines of the phosphodiester backbone in the 750-850 cm-1 region. A conformation-sensitive guanine mode, which yields Raman lines near 682, 668, or 625 cm-1 in B (C2'-endo, anti), A (C3'-endo, anti) or Z (C3'-endo, syn) structures, respectively, is the most useful for quantitative analysis. In D2O, the guanine line of Z-DNA is shifted to 615 cm-1, permitting its detection even in the presence of proteins.     

Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.     

Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin.

The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5).     

Resonance Raman spectroscopy of horseradish peroxidase derivatives and intermediates with excitation in the near ultraviolet

Resonance Raman enhancement of derivatives and intermediates of horseradish peroxidase in the near ultraviolet (N-band excitation) results in intensity and enhancement patterns that are different from those normally observed within the porphyrin Soret (B-band) and alpha-beta (Q-band) absorptions. In particular it allows the resolution of resonance Raman spectra of horseradish peroxidase compound I. The bands above 1300 cm-1 can be assigned to porphyrin vibrational modes that are characteristically shifted in frequency due to removal of an electron from the porphyrin ring. The resonance Raman frequency shifts follow normal mode compositions. Relative to resonance Raman spectra of compound II, the v4 frequency (primarily Ca-N) exhibits a 20 cm-1 downshift. The v2, v11, and v37 vibrational frequencies whose mode compositions are primarily porphyrin Cb-Cb, exhibit 10-20 cm-1 upshifts. The v3, v10, and v28 frequencies, whose mode compositions are primarily Ca-Cm, exhibit downshifts. The downshifts for v3 and v10 are small, 3-5 cm-1; however, the downshift for v28 is 14 cm-1. These frequency shifts are consistent with those of previously published resonance Raman studies of model compounds. In contrast to reports from other laboratories, the data presented here for horseradish peroxidase compound I can be attributed unambiguously to resonance Raman scattering from a porphyrin pi-cation radical.     

Resonance Raman spectroscopy of polynucleotides

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.     

Characteristics and variations of B-type DNA conformations in solution: a quantitative analysis of Raman band intensities of eight DNAs

Raman spectra were obtained from four bacterial DNAs varying in GC content and four periodic DNA polymers in 0.1 M NaCl at 25 degrees C. A curve fitting procedure was employed to quantify and compare Raman band characteristics (peak location, height, and width) from 400 to 1600 cm-1. This procedure enabled us to determine the minimum number of Raman bands in regions with overlapping peaks. Quantitative comparison of the Raman bands of the eight DNAs provided several new results. All of the DNAs examined required bands near 809 (+/- 7) and 835 (+/- 5) cm-1 to accurately reproduce the experimental spectra. Since bands at these frequencies are associated with A-family and B-family conformations, respectively, this result indicates that all DNAs in solution have a mixture of conformations on the time scale of the Raman scattering process. Band characteristics in the 800-850-cm-1 region exhibited some dependence on CG content and base pair sequence. As previously noted by Thomas and Peticolas [Thomas, G. A., & Peticolas, W. L. (1983) J. Am. Chem. Soc. 105, 993], the poly[d(A)].poly[d(T)] spectra were qualitatively distinct in this region. The A-family band is clearly observed at 816 cm-1. The intensity of this band and that of the B-family band at 841 cm-1 were similar, however, to intensities in the natural DNA spectra. Three bands at 811, 823, and 841 cm-1 were required to reproduce the 800-850-cm-1 region of the poly[d(A-T)].poly[d(A-T)] spectra. This may indicate the presence of three backbone conformations in this DNA polymer. Analysis of intensity vs. GC content for 42 Raman bands confirmed previous assignments of base and backbone vibrations and provided additional information on a number of bands.     

Resonance Raman studies of the HOOP modes in octopus bathorhodopsin with deuterium-labeled retinal chromophores

Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm-1 for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm-1 to C10H and C14H HOOP modes, and the band at 940 cm-1 to C11H = C12H Au-like HOOP mode. Except for a 26 cm-1 downward shift, the C11H = C12H Au-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C10H and C14H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C7H and C8H HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C11H = C12H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C11 = C12 double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C11H = C12H Au-like HOOP mode. The twisted nature of the chromophore, semiquantitatively discussed here, likely affects the lambda max of the chromophore and its enthalpy.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.     

Resonance Raman enhancement of phenyl ring vibrational modes in phenyl iron complex of myoglobin.

Resonance Raman spectra are reported for the organometallic phenyl-FeIII complexes of horse heart myoglobin. We observed the resonance enhancement of the ring vibrational modes of the bound phenyl group. They were identified at 642, 996, 1,009, and 1,048 cm-1, which shift to 619, 961, 972, and 1,030 cm-1, respectively, upon phenyl 13C substitution. The lines at 642 and 996 cm-1 are assigned, respectively, as in-plane phenyl ring deformation mode (derived from benzene vibration No. 6a at 606 cm-1) and out-of-plane CH deformation (derived from benzene vibration No. 5 at 995 cm-1). The frequencies of the ring "breathing" modes at 1,009 and 1,048 cm-1 are higher than the corresponding ones in phenylalanine (at 1,004 and 1,033 cm-1) and benzene (at 992 and 1,010 cm-1), indicating that the ring C--C bonds are strengthened (or shortened) when coordinated to the heme iron. The excitation profiles of these phenyl ring modes and a porphyrin ring vibrational mode at 674 cm-1 exhibit peaks near its Soret absorption maximum at 431 nm. This appears to indicate that these phenyl ring modes may be enhanced via resonance with the Soret pi-pi transition. The FeIII--C bond stretching vibration has not been detected with excitation wavelengths in the 406.7-457.9-nm region.     

Structure of DNA hydration shells studied by Raman spectroscopy

We have used Raman scattering to study the water O-H stretching modes at approximately 3450 and approximately 3220 cm-1 in DNA films as a function of relative humidity (r.h.). The intensity of the 3220-cm-1 band vanishes as the r.h. is decreased from 98% to around 80%, which indicates that the hydrogen-bond network of water is disrupted in the primary hydration shell (which therefore cannot have an "ice-like" structure). The number of water molecules in the primary hydration shell was determined from the intensity of the approximately 3200-cm-1 band as about 30 water molecules per nucleotide pair. The approximately 3400-cm-1 O-H stretch band was used for determining the total water content, and this band persists at 0% r.h., implying that 5-6 tightly bound water molecules per nucleotide pair remain. The frequency of the approximately 3400-cm-1 O-H stretch mode is lower by 30 to 45 cm-1 in the primary hydration shell compared to free water. The water content as a function of r.h. obtained from these experiments agrees with gravimetric measurements. The disappearance of the approximately 3200-cm-1 band and the shift of the approximately 3400-cm-1 O-H stretch band provide a reliable way of measuring the hydration number of DNA.     

Metal-ligand vibrations of cyanoferric myeloperoxidase and cyanoferric horseradish peroxidase: evidence for a constrained heme pocket in myeloperoxidase

The low-frequency FeCN vibrations of cyanoferric myeloperoxidase (MPO) and horseradish peroxidase (HRP) have been measured by resonance Raman spectroscopy. The ordering of the frequencies of the predominantly FeC stretching and FeCN bending normal vibrational modes in the two peroxidases differs. These normal mode vibrations are identified by their wavenumber shifts upon isotopic substitution of the cyanide ligand. For MPO, the stretching mode nu 1 (361 cm-1) occurs at a lower frequency than the bending mode delta 2 (454 cm-1). For HRP, the order is reversed as nu 1 (456 cm-1) is at a higher frequency than delta 2 (404 cm-1). Normal coordinate analyses and model complexes have been used to address the origin of this behavior. The nu 1 stretching frequencies in cyanide complexes of iron porphyrin and iron chlorin model compounds are similar to one another and to that of HRP. Thus, the inverted order and altered frequencies of the nu 1 and delta 2 vibrations in MPO, relative to those in HRP and the model compounds, are not inherent to the proposed iron chlorin prosthetic group in MPO but, rather, are attributed to distinct distal environmental effects in the MPO active site. The normal coordinate analyses for MPO and HRP showed that the nu 1 and delta 2 vibrational frequencies are not pure; the potential energy distributions for these modes respond not only to the geometry but also to the force constants of the nu(FeC) and delta(FeCN) internal coordinates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonant and localized breathing modes in terminal regions of the DNA double helix.

A Green's function approach is used in constructing a dynamic model of a semi-infinite length of the DNA homopolymer B poly(d) . poly(d). Considerable attention is focused on the hydrogen bond stretching close to the terminus. A melting (or breathing) coordinate (M) is defined as an average over the three linking hydrogen bond stretches in a unit cell. The thermal mean squared amplitude of (M) is enhanced at the chain end compared with the interior. Spectral branches at 69, 80 and 105 cm-1, as well as a local mode at 75 cm-1, are primary contributors to the enhancement. We suggest that this fact can affect the thermal melting of a DNA double helical homopolymer, enhancing the tendency to start from an end (if one is available). We show how certain infinite chain modes with small (M) amplitude can turn into breathing modes near the terminus, and suggest that the same phenomenon may occur near other specific base-pair sequences. There is also considerable attention paid to the low microwave region from approximately 0 to 1.75 cm-1. The thermally activated modes in this frequency region contribute approximately (0.02 A)2 to [M2(0)] at 40 K, approximately two orders of magnitude greater than for [M2(infinity)]. Most important however, is the existence of narrow resonant modes in this frequency region. Particularly pronounced resonances near 0.03 cm-1 and 0.08 cm-1 (approximately 0.9 and 2.4 GHz) amplify M2(0) at the terminus by about for orders of magnitude over the infinite chain value M2(infinity).     

Raman spectroscopy of interferon-induced 2',5'-linked oligoadenylates

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.     

Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.     

Resonance Raman spectroscopy of complexes of the helix destabilizing proteins GP32 and GP5 with poly(rA) and poly(dA)

The bacteriophage T4 helix destabilizing protein (hdp) gp32 and its complexes with poly(rA) and poly(dA) were studied with ultra-violet resonant Raman spectroscopy. The UV-resonant Raman (UV-RR) spectrum of the complex of gp5, the coat protein of bacteriophage M13, with poly(dA) was also measured and is compared with the spectrum of the gp 32/poly(dA) complex. The excitation wavelength was 245.1 nm. This is on the far UV-side of the first absorption bands of adenine and near a "window" in the protein absorption spectrum. The overlap of fluorescence due to chromophores present in the protein and resonance Raman scattering was prevented by this choice of wavelength. The spectra of the protein/polynucleotide complexes are compared with the native nucleotide spectra measured at varying temperatures. The hyperchromicity which is expected when a nucleotide changes from a stacked to an unstacked conformation was not observed for poly(rA), neither upon temperature increase nor on protein binding. In both cases poly(dA) revealed a clear hyperchromicity. This different behavior of poly(rA) and poly(dA) is probably a consequence of their different conformations. The contributions of the proteins to the spectra is weak except for two bands, at 1550 and 1610 cm-1 due to tryptophan (in case of gp32) and one band near 1610 cm-1 due to tyrosine and phenylalanine.     

63/65Cu and 1/2H2O isotope shifts in the low-temperature resonance Raman spectrum of fungal laccase

Resonance Raman spectra are reported for the type 1 Cu site of fungal laccase at 295 and 77 K. The low-temperature spectra show enhanced resolution and reveal several weak bands not previously observed, as well as overtone and combination bands associated with the strong approximately equal to 400 cm-1 fundamentals. A novel low-temperature Raman difference technique has been used to obtain 63/65Cu and 1/2H2O isotope shifts. The strong band at 428 cm-1, and the moderate intensity bands at 408 and 387 cm-1 show small (under 0.6 cm-1 63/65Cu isotope shifts. The aggregate shift is substantially less than that expected for an isolated Cu-S(cys) stretch, implying a high degree of mixing of this coordinate with internal modes of the ligands. 1/2H2O shifts of 1.1 and approximately equal to 0.3 cm-1 are observed for the 387 and 428 cm-1 bands. The isotope shift patterns are quite similar for fungal and tree laccase, as are the frequencies of the dominant bands, indicating that the large differences in relative intensity are primarily associated with differences in the excited state potential. The frequency and isotope shift patterns are appreciably different, however, from those observed for azurin and stellacyanin. In contrast to the other 'blue' Cu proteins, fungal laccase shows no moderate intensity band near 270 cm-1 which can be associated with Cu-imidazole stretching; weak features are seen in this region, but the intensities are too low to determine their 1/2H2O sensitivity. The C-S stretching mode of fungal laccase is identified at 737 cm-1, shifting to 741 cm-1 at 77 K. It is about 10 cm-1 lower than for most 'blue' Cu proteins, and the difference is suggested to reflect smaller kinematic coupling between the C-S and Cu-S coordinates, associated with a smaller Cu-S-C angle. Combination modes of the approx. 400 cm-1 fundamentals are substantially stronger, relative to the overtones, than is predicted by first-order scattering theory, implying changes in the excited-state normal modes (Dushinsky effect) associated with force constant alterations.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.