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.     

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.     

Flexibility of DNA and RNA upon Binding to Different Metal Cations. An Investigation of the B to A to Z Conformational Transition by Fourier Transform Infrared Spectroscopy

Abstract

The interaction of DNA and RNA with Cu(II), Mg(II), [Co(NH₃)₆]³⁺ [Co(NH₃)₅Cl]²⁺ chlorides and, cis- and trans-Pt(NH₃)₂Cl₂ (CIS-DDP, trans-DDP) has been studied by Fourier Transform Infrared (FT-IR) spectroscopy and a correlation between metal-base binding and conformational transitions in the sugar pucker has been established. It has been found that RNA did not change from A-form on complexation with metals, whereas DNA exhibited a B to Z transition. The marker bands for the A-form (C′₃-endo-anti conformation) were found to be near 810–816 cm^?1, while the bands at 825 and 690 cm^?1 are marker bands for the B- conformation (C′₂-endo, anti), The B to Z (C₃′-endo, syn conformation) transition is characterized by the shift of the band at 825 cm^?1 to 810–816 cm^?1 and the shift of the guanine band at 690 cm^?1 to about 600–624 cm^?1.     

Vibrational Circular Dichroism Spectra of Proteins in the Amide III Region: Measurement and Correlation of Bandshape to Secondary Structure

Vibrational circular dichroism (VCD) spectra have been measured for 23 globular proteins dissolved in H₂O/phosphate buffer over the 1400 to 1100 cm⁻¹region which encompasses the amide III mode. Spectral responses characteristic of the dominant secondary structure type were found as broad features at ∼1300 cm⁻¹, with the extreme forms having positive VCD for highly helical proteins and negative VCD for highly sheet-containing proteins. Quantitative correlation with secondary structure was carried out using previously developed factor analysis and restricted multiple regression (FA/RMR) techniques. Since the absorbance intensity of the amide III mode is difficult to determine due to overlap with other transitions, an alternative, absolute intensity-independent, simple structural analysis method was used. A linear regression was developed between the fractional components of secondary structure for the protein set and the overlap integrals of the normalized spectra from the set with that of a selected protein. The results of this simple method are quite comparable to those of the FA/RMR approach for analysis with amide III VCD. On the other hand, test calculations with the new method when used with electronic CD spectra are not as good as FA/RMR due to its more intensity-dependent relationship with secondary structure.     

VCD spectroscopic properties of the β‐hairpin forming miniprotein CLN025 in various solvents

Electronic and vibrational circular dichroism are often used to determine the secondary structure of proteins, because each secondary structure has a unique spectrum. Little is known about the vibrational circular dichroic spectroscopic features of the β‐hairpin. In this study, the VCD spectral features of a decapeptide, YYDPETGTWY (CLN025), which forms a stable β‐hairpin that is stabilized by intramolecular weakly polar interactions and hydrogen bonds were determined. Molecular dynamics simulations and ECD spectropolarimetry were used to confirm that CLN025 adopts a β‐hairpin in water, TFE, MeOH, and DMSO and to examine differences in the secondary structure, hydrogen bonds, and weakly polar interactions. CLN025 was synthesized by microwave‐assisted solid phase peptide synthesis with N^α‐Fmoc protected amino acids. The VCD spectra displayed a (?,+,?) pattern with bands at 1640 to 1656 cm^?1, 1667 to 1687 cm^?1, and 1679 to 1686 cm^?1 formed by the overlap of a lower frequency negative couplet and a higher frequency positive couplet. A maximum IR absorbance was observed at 1647 to 1663 cm^?1 with component bands at 1630 cm^?1, 1646 cm^?1, 1658 cm^?1, and 1675 to 1680 cm^?1 that are indicative of the β‐sheet, random meander, either random meander or loop and turn, respectively. These results are similar to the results of others, who examined the VCD spectra of β‐hairpins formed by ^DPro‐Xxx turns and indicated that observed pattern is typical of β‐hairpins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 442–450, 2010. 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     

Studies on photosynthetic oxygen-evolving complex by means of Fourier transform infrared spectroscopy: calcium and chloride cofactors

Structural roles of functional Ca²⁺ and Cl⁻ ions in photosynthetic oxygen-evolving complexes (OEC) were studied using low- (640–350 cm⁻¹) and mid- (1800–1200 cm⁻¹) frequency S₂/S₁ Fourier transform infrared (FTIR) difference spectroscopy. Studies using highly active Photosystem (PS) II core particles from spinach enabled the detection of subtle spectral changes. Ca²⁺-depleted and Ca²⁺-reconstituted particles produced very similar mid- and low-frequency spectra. The mid-frequency spectrum was not affected by reconstitution with ⁴⁴Ca isotope. In contrast, Sr²⁺-substituted particles showed unique spectral changes in the low-frequency Mn–O–Mn mode at 606 cm⁻¹ as well as in the mid-frequency carboxylate stretching modes. The mid-frequency spectrum of Cl⁻-depleted OEC exhibited marked changes in the carboxylate stretching modes and the suppression of protein modes compared with that of Cl⁻-reconstituted OEC. However, Cl⁻-depletion did not exert significant effects on the low-frequency spectrum.     

The detection of food soils on stainless steel using energy dispersive X-ray and Fourier transform infrared spectroscopy

Organic soiling is a major issue in the food processing industries, causing a range of biofouling and microbiological problems. Energy dispersive X-ray (EDX) and Fourier transform infra red spectroscopy (FT-IR) were used to quantify and determine the biochemical groups of food soils on stainless steel surfaces. EDX quantified organic material on surfaces where oily based residues predominated, but was limited in its usefulness since other food soils were difficult to detect. FT-IR provided spectral ‘fingerprints’ for each of the soils tested. Key soiling components were associated with specific peaks, viz. oils at 3025 cm^?1–3011 cm^?1, proteins at 1698 cm^?1–1636 cm^?1 and carbohydrates at 1658 cm^?1–1596 cm^?1, 783 cm^?1–742 cm^?1. High concentrations of some soils (10%) were needed for detection by both EDX and FT-IR. The two techniques may be of use for quantifying and identifying specific recalcitrant soils on surfaces to improve cleaning and hygiene regimes.     

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.     

Absolute configuration determination and conformational analysis of (−)‐(3S,6S)‐3α,6β‐diacetoxytropane using vibrational circular dichroism and DFT techniques

The absolute configuration of semisynthetic (?)‐3α,6β‐acetoxytropane 1 , prepared from (?)‐6β‐hydroxyhyoscyamine 2 , has been determined using vibrational circular dichroism (VCD) spectroscopy. The vibrational spectra (IR and VCD) were calculated using DFT at the B3LYP/DGDZVP level of theory for the eight more stable conformers which account for 99.97% of the total relative abundance in the first 10 kcal/mol range. The calculated VCD spectra of all considered conformations showed two distinctive spectral ranges, one between 1300 and 1200 cm^?1, and the other one in the 1150–950 cm^?1 region. When compared with the experimental VCD spectrum, the first spectral region confirmed the calculated conformational preferences, whereas the second region showed little change with conformation, thus allowing the determination of the absolute configuration of 1 as (3S,6S)‐3α,6β‐diacetoxytropane. Also, the bands in the second region showed similarities between 1 and 2 in both the experimental and calculated VCD spectra, suggesting that these bands are mainly related to the absolute configuration of the rigid tropane ring system, since they show conformational independency, no variations with the nature of the substituent, and are composed by closely related vibrational modes. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

CD and Fourier transform ir spectroscopic studies of peptides. II. Detection of β-turns in linear peptides

Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) β-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of β-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the β-turn band shewed up between 1639 and 1633 cm^?1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band war-also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3₁₀-helical polypeptides and protein in D₂O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508–512; H. H. Mantsch, A. Perczel. M. Hollósi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch. A. Perczel, M. Hollósi, and G. Fasman (1992), Biopolymers. Vol. 33, pp. 201–207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Tedd, and G. L. Millhauser (1992). Nature, Vol. 359, pp. 653–655], suggest that the amide I band, with a major contribution from the acceptor C ? O of the 1 ← 4 intramolecular H bond of β-turns, appears near or below 1640 cm^?1, rather than above 1660 cm^?1. In TFE, bands between 1670 and 1660 cm^?1 are mainly due to “free” carbonyls, that is, C ? O's of amides that are solvated but not involved in the characteristic H bonds of periodic secondary structures or β-turns. © 1994 John Wiley & Sons, Inc.     

Direct discrimination of different plant populations and study on temperature effects by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to characterise highland and lowland populations of Polygonum minus Huds. grown in different controlled environments. A thermal perturbation technique of two-dimensional correlation infrared spectroscopy (2D-IR) correlation spectra was applied to establish differences between the populations. The absorption peaks at 3,480 cm^?1 (hydroxyl group), 2,927 cm^?1 (methyl group), 1,623 cm^?1 (carbonyl group), and 1,068 cm^?1 (C–O group) were particularly powerful in separating the populations. These peaks, which indicate the presence of carbohydrate, terpenes, amide and flavonoids were more intense for the highland populations than lowland populations, and increased in environments with a higher temperature. Wavenumbers (1,634, 669 cm^?1) and (1,634, 1,555 cm^?1) in the 2D-IR correlation spectra provided fingerprint signals to differentiate plants grown at different temperatures. This study demonstrates that IR fingerprinting, which combines mid-IR spectra and 2D-IR correlation spectra, can directly discriminate different populations of P. minus and the effects of temperature.     

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.     

Infrared spectrum of a DNA-RNA hybrid

The infrared absorption spectrum in the 4000–400 cm^? region of an oriented film of a DNA–RNA hybrid in its undeuterated and deuterated states was observed with the polarized radiation. Most of the stronger bands found in the double-helical DNA's and double-helical RNA's are identified in the spectrum of the hybrid. The absorption band at 1225 cm^?1 shows a perpendicular dichroism and that at 1085 cm^?1 shows almost no dichroism. These facts indicate that the orientation of the group with respect to the helix axis in the hybrid structure is not entirely the same as that in the double-helical Na DNA at, 75% RH., although the x-ray diffraction pattern of the hybrid is quite similar to that of the DNA A form. The PO₂^? orientation is not the same as that in the double-helical RNA either. The observed dichroism is explained, however, by considering that the PO₂^? group in the RNA moiety takes nearly the same orientation as that in the double-helical RNA, and the PO₂^? group in the DNA moiety takes nearly the same as that in the double-helical DNA.     

Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies

The ir spectra of lathyritic rat skin collagen and calf skin gelatin solutions at a variety of temperatures were obtained using Fourier transform ir spectroscopy and a 9-reflection, 2-pass ZnSe prism sample cell. The spectra were then deconvolved (based on Kauppinnen's method) and the behavior of the amide I band at ～ 1650 cm^?1 observed in detail. Throughout the temperature range studied (4–50°C), three component absorption peaks within the amide I band (at 1633, 1643, and 1660 cm^?1) are common to the spectra irrespective of the degree of triple helix content of the sample. Changes in the relative intensities of these component peaks are, however, conformationally dependent. During denaturation of the triple helix, the dominant 1660-cm^?1 component in the native collagen spectrum diminishes and the 1633-cm^?1 peak becomes relatively intensified. The inherently strong basicity of the carbonyl group of the proline residues together with the frequent occurrence of this imino acid in the X position of the Gly-X-Y triplet of collagen largely accounts for the ?30-cm^?1 shift of the amide I band during denaturation. Temperature and conformationally dependent changes in the fine structure of the amide I band from dilute solutions of collagen can be monitored in a reproducible and quantitative fashion.     

Infrared and Raman spectroscopy of carbohydrates. Part VI: Normal coordinate analysis of V-amylose

A normal coordinate analysis of V-amylose has been performed for an isolated 6₁ helical chain. Negligible splitting from interactions of vibrations of successive residues is expected between A and E vibrational species due to the large size of the monomer unit. As a result, calculation of only the totally symmetric A modes represents an adequate approximation to the vibrational spectrum of helical polysaccharides. Using this method together with a valence force field we have obtained good agreement between the observed and calculated frequencies. In addition, the computed potential energy distribution and Cartesian displacement coordinates match previous experimental assignments, based on deuterium exchange. The analysis also supports the proposed mechanism for conversion of V-amylose to the more extended B-form. This conversion results in an observed frequency shift for the Raman line at 946 cm^?1 which is predicted by the calculations.     

Raman spectroscopic study on low-frequency collective modes in self-associates of guanosine monophosphates

Low-frequency Raman spectra of the self-associates of guanosine monophosphates (GMPs) Na₂ · 5′GMP, K₂ · 5′GMP, Na₂ · 3′GMP, and K₂ · 3′GMP, and polyribonucleic acid K · poly(rG), were obtained. In acidic gels and dried fibers, GMP molecules are known to form helical stacks of hydrogen-bonded tetramers. Some low-frequency collective modes specific to the helically stacked structures were observed. We examined the dependence of these modes on counterions and water content. The lowest frequency mode at ca. 20 cm^?1 is sensitive to the water content of the sample and is clearly visible in solid-state samples, so it works as a marker band of the environmental condition of the helices. The intensity and the peak frequency of this mode in solid-state samples depend on the helical structure and counterions. The broad peaks in the vicinity of 100 cm^?1 are influenced by cations and are independent of water content.     

Vibrational and chiroptical spectroscopic characterization of γ‐turn model cyclic tetrapeptides containing two β‐Ala residues

The optical spectroscopic characterization of γ‐turns in solution is uncertain and their distinction from β‐turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on γ‐turn model cyclic tetrapeptides cyclo(Ala‐β‐Ala‐Pro‐β‐Ala) ( 1 ), cyclo(Pro‐β‐Ala‐Pro‐β‐Ala) ( 2 ) and cyclo(Ala‐β‐Ala‐Ala‐β‐Ala) ( 3 ). Conformational analysis performed at the 6‐31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1‐3 , featuring two inverse γ‐turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n→π* band near 230 nm and a positive π→π* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1‐3 show similar spectra with blue‐shifted bands. The VCD spectra in ACN‐d₃ of 1 and 2 show a +/?/+/? amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2 . A ?/+/+/? amide I VCD pattern was measured for 3 in TFE‐d₂. All three peptides give a positive couplet or couplet‐like feature (+/?) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at ～1630 cm^?1 or below) are indicative of a C₇ H‐bonded inverse γ‐turns with Pro in position 2, while γ‐turns encompassing Ala absorb at higher frequency (above 1645 cm^?1). Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

A vitrational study of poly (L-proline) I and II in the far infrared.

The far infrared spectra of poly(L -proline) I (190–35 cm^?1) and II (400–35 cm^?1) were obtained in the solid state at both 300° and 110°K. A significant difference in the region below 100 cm^?1 was observed. A very intense band located at 60 cm^?1 in the infrared spectrum of form II has no counterpart in form I. This indicates the sensitivity of low-frequency vibrations to the difference in conformation assumed by both forms in the solid state. Additional bands observed in this study are correlated with ir and Raman data previously reported and tentative assignments are made using the results of normal mode calculations (in the single-chain approximation) which have been reported.     

Complexes of (dG-dC)20 with Mn2+ Ions: A Study by Vibrational Circular Dichroism and Infrared Absorption Spectroscopy

Abstract

The B-Z transition of the synthetic oligonucleotide, (dG-dC)₂₀, induced by Mn²⁺ ions at room temperature, was investigated by absorption and Vibrational Circular Dichroism (VCD) spectroscopy in the range of 1800–800 cm^?1. Metal ion concentration was varied from 0 to 0.73 M Mn²⁺ (0 to 8.5 moles of Mn²⁺ per mole of oligonucleotide phosphate, [Mn]/[P]). While both types of spectra showed considerable changes as the Mn²⁺ concentrations were raised, differences between the two were often complementary in their expression and extent, those displayed by VCD being more clearly evident due to the inversion of the opposite helical sense from the right-handed to the left-handed conformation. The main phase of the transition occurred in the metal ion concentration between 0.8?1.1 [Mn]/[P]. Gradual changes that took place in the spectra were interpreted in terms of simultaneous processes that depended on metal ion concentration, namely B-Z transformation, binding of Mn²⁺ to phosphates and to nitrogen bases, and partial denaturation. Below～0.6 [Mn]/[P], only a small portion of the oligonucleotide adopted the Z conformation within a 3 hour period, whereas conversion was completed in the same time interval for concentrations between 0.9?1.2 [Mn]/[P]. At [Mn]/[P] > 1.7, complete transition to the Z-form took place immediately on adding Mn²⁺. Applying VCD spectroscopy in combination with conventional infrared absorption proved most useful for corroborating changes in the absorption spectra, and for detecting in an unique manner, not attainable by absorption methods, conformational changes that lead to the inversion of the helical sense of the oligonucleotide.     

Fourier transform infrared spectroscopy of aqueous dispersions of phosphatidylserine-cholesterol mixtures

The effect of cholesterol on vibrational spectra in the non polar and in the polar region of dimyristoyl phosphatidylserine (DMPS) and of phosphatidylserine from bovine spinal cord (PS) has been investigated. The small shifts in the methylene CH stretching frequencies after taking into account the contribution of the cholesterol spectrum were interpreted as a combined effect of cholesterol on the conformation of the chains and of the lesser contributions of the cholesterol methyl groups. Cholesterol also influences the ratio of the trans (1465 cm^–1) to the lower wavelength (1457 cm^–1) CH₂ bending bands. No significant direct effect of cholesterol on the vibration of the polar residues was discerned. The small shift of the carboxylate band observed below the phase transition is probably due to the change in the intermolecular zwitterions when the average distance between the neighboring polar groups increases due to incorporation of cholesterol molecules.Abbreviations PS phosphatidylserine natural - DMPS dimyristoyl phosphatidylserine - DPPC dipalmitoyl phosphatidylcholine - FTIR Fourier transform infrared spectroscopy - DSC differential scanning calorimetry - PE phosphatidylethanolamine Offprint requests to: D. Bach