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.     

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.     

DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

Vibrational circular dichroism of A-, B-, and Z-form nucleic acids in the PO2-stretching region.

Vibrational circular dichroism (VCD) spectra were measured for H2O solutions of several natural and model DNAs (single and double strands, oligomers and polymers) in the B-form, poly(dG-dC)-poly(dG-dC) in the Z-form, and various duplex RNAs in an A-form over the PO2-stretching region. Only the symmetric PO2 stretch at approximately 1075 cm-1 yields a significant intensity VCD signal. Differences of the PO2-stretching VCD spectra found for these conformational types are consistent with the spectral changes seen in the base region, but no sequence dependence was seen in contrast to VCD for base modes. The B to Z transition is accompanied by an inversion of the PO2- VCD spectra, which is characteristic of the change in the helical sense of the nucleic acid backbone. A-RNAs give rise to the same sense of couplet VCD as do B-DNAs but have a somewhat different shape because of overlapping ribose modes. These PO2- VCD spectral characteristics have been successfully modeled using simple dipole coupling calculations. The invariability of the symmetric PO2- stretching mode VCD spectra to the base sequence as opposed to that found for the C = O stretching and base deformation modes is evidence that this mode will provide a stable indication of the DNA helical sense.     

Phase equilibrium in poly(rA).poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).     

Helical nature of poly(dI-dC).poly(dI-dC). Vibrational circular dichroism results.

Poly(dI-dC).poly(dI-dC) was studied using vibrational circular dichroism and IR spectroscopy in both the base deformation C = O and symmetric PO2- stretching regions. VCD spectra of this duplex under low salt conditions are consistent with its having a B-form structure. Addition of 5 M NaCl leads to relatively uniform VCD intensity loss which is consistent with loss of helical structure rather than formation of an intermediate state between the B and Z forms. This duplex polymer under high salt conditions with added NiCl2 shows aggregation effects, but its IR and VCD spectra have characteristic features of the Z-form DNA conformation. The cooperative change of backbone and base pair structure upon thermal denaturation is indicated by the simultaneous collapse of the VCD at 65 degrees C in both the PO2- and C = O stretching regions. This study further demonstrates that the VCD bandshape of a specific localized nucleic acid vibrational transition can be a useful indicator of the helical handedness. The empirical conformational interpretations are supported by simulated VCD spectra, which are in excellent agreement with the experimental results, based on dipole coupling calculations.     

The stability of DNA-porphyrin complexes in the presence of Mn(II) ions

The complex formation of porphyrins with DNA leads to changes of stability of DNA. In the present study we investigated binding properties and the thermodynamic parameters of a water-soluble, cationic planar Cu(II)-containing meso-tetrakis(4-N-butyl-pyridiniumyl)porphyrin [CuTButPyP4] and nonplanar Co(II)-containing meso-tetrakis(4-N-butyl-pyridiniumyl)porphyrin [CoButPyP4] with calf thymus DNA in the presence of divalent manganese ions. For displaying the changes of thermodynamic parameters (T_m and ΔT) the melting curves of DNA-porphyrin complexes in the presence of Mn²⁺ ions have been obtained. The enthalpy (ΔH) of helix-coil transition has been also evaluated. It was shown that the binding of ions to DNA proceeds in two stages depending on the manganese/DNA phosphates molar ratio [Mn]/[P]. At the first stage (0.001 < [Mn]/[P] < 1), the interaction of manganese ions with DNA phosphates occurs, causing an additional screening of their negative charge and the stabilization of the double helix. As a result, the best conditions for intercalation of CuTButPyP4 or of peripheral rings of CoButPyP4 occur. The significant increase of T_m, but less changes of ΔT were observed. At the second stage (1 < [Mn]/[P] < 4), the ions interact with both the phosphates and the nitrogen bases of DNA. At this stage, it is possible for the manganese ion to coordinate simultaneously to the oxygen atom of the phosphate and the neighboring base of DNA. At a higher [Mn]/[P] ratio, the destabilization of the double helix begins, and partial breakage of the hydrogen bonds between the nitrogen bases occurs. Respectively the destabilization of DNA in the presence of both porphyrins takes place.     

菠萝叶片PEP羧激酶与底物结合时构象的变化

菠萝叶片PEP羧激酶与底物OAA和ATP及配基Mn~(2+)等结合时引起紫外差示吸收光谱峰位和方向上的变化。OAA与酶结合诱导产生的差示吸收光谱在268—280nm处有一个宽负峰。ATP与酶结合出现两个差示负峰(242.5和273.5nm)。双底物OAA和ATP同时与酶结合,光谱上呈现252nm和268nm两个峰。Mn~(2+)不论与ATP或与ATP+OAA一起与酶反应时,皆使原来的峰位漂移,且正负方向逆转。酶蛋白在323nm有最大的荧光发射。OAA引起荧光发射强度增大,ATP及ATP+Mn~(2+)则减弱荧光发射。Mn~(2+)与OAA及ATP的复合效应导致荧光强度进一步减弱。     

Poly(rA).poly(rU) with Ni(2+) ions at different temperatures: infrared absorption and vibrational circular dichroism spectroscopy

Phase transitions were studied of the sodium salt of poly(rA).poly(rU) induced by elevated temperature without Ni(2+) and with Ni(2+) in 0.07 M concentration in D(2)O (approximately 0.4 [Ni]/[P]). The temperature was varied from 20 degrees C to 90 degrees C. The double-stranded conformation of poly(rA).poly(rU) was observed at room temperature (20 degrees C-23 degrees C) with and without Ni(2+) ions. In the absence of Ni(2+) ions, partial double- to triple-strand transition of poly(rA).poly(rU) occurred at 58 degrees C, whereas only single- stranded molecules existed at 70 degrees C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni(2+) ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45 degrees C and 70 degrees C with maximum stability around 53 degrees C. Triple- to single-stranded transition of poly(rA).poly(rU) occurred around 72 degrees C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86 degrees C. Hysteresis took place in the presence of Ni(2+) during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO₂ stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.     

Poly(rA) • Poly(rU) with Ni2+ Ions at Different Temperatures: Infrared Absorption and Vibrational Circular Dichroism Spectroscopy

Abstract

Phase transitions were studied of the sodium salt of poly(rA) ?poly(rU) induced by elevated temperature without Ni²⁺ and with Ni²⁺ in 0.07 M concentration in D₂O (～0.4 [Ni]/[P]). The temperature was varied from 20° C to 90° C. The double-stranded conformation of poly(rA)?poly(rU) was observed at room temperature (20° C—23° C) with and without Ni²⁺ ions. In the absence of Ni²⁺ ions, partial double- to triple-strand transition of poly(rA) ?poly(rU) occurred at 58° C, whereas only single-stranded molecules existed at 70° C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni²⁺ ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45° C and 70° C with maximum stability around 53° C. Triple-to single-stranded transition of poly(rA) ?poly(rU) occurred around 72° C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86° C. Hysteresis took place in the presence of Ni²⁺ during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

Spectroscopic investigations of the structure of DNA complexes with Mn2+ in UV and IR regions

Based on the data of UV and IR spectroscopy, electronic and vibrational circular dichroism, the interaction of manganese ions with DNA was investigated. It was shown that the binding of ions to DNA proceeds in three stages depending on the manganese-to-DNA phosphates molar ratio [Mn]/[P]. At the first stage ([Mn]/[P] < or = 1), the interaction of manganese ions with DNA phosphates occurs, causing a partial screening of their negative charge and the stabilization of the double helix. At the second stage (1 < [Mn]/[P] < 6), the ions interact with both the phosphates and the nitrogen bases of DNA. At this stage, it is possible for the manganese ion to coordinate simultaneously to the oxygen atom of the phosphate and the neighbouring base of DNA. At a higher [Mn]/[P] ratio, the destabilization of the double helix begins, and partial breakage of the hydrogen bonds between the nitrogen bases occurs.     

Discriminating 3(10)- from alpha-helices: vibrational and electronic CD and IR absorption study of related Aib-containing oligopeptides

Model peptides based on -(Aib-Ala)(n)-, and (Aib)(n)-Leu-(Aib)(2) sequences, which have varying amounts of 3(10)-helical character, were studied by use of vibrational and electronic circular dichroism (VCD and ECD) and Fourier transform infrared (FTIR) absorption spectroscopies to test the correlation of spectral response and conformation. The data indicate that these peptides, starting from a length of about four to six residues, predominantly adopt a 3(10)-helical conformation at room temperature. The longest model peptides, depending on the series, may evidence some alpha-helical contribution to the spectra, while the shorter ones, with less than six residues, have much less order. The IR absorption spectra (as supported by theory) showed only small frequency changes between 3(10)- and alpha-helices. By contrast, solvent effects are a source of much bigger perturbations. The ECD results show that the intensity ratio for the approximately 222-nm to approximately 208-nm bands, while useful for distinguishing between these two helical types in some sequences, may have a narrower range of application than VCD. However, the VCD data presented here continue to support the proposed discrimination between alpha- and 3(10)-helices based on qualitative amide I and II bandshape differences. The present study shows the intensities of the 3(10)-helical amide I (peak-to-peak) to its amide II VCD to be of the same order and useful for discriminating them from alpha-helices, whose amide I dominates the amide II in intensity. This qualitative result is experimentally independent of the amount of alphaMe-substituted residues in the sequence. These experimental VCD results are consistent in detail with theoretical spectral simulations for Ac-(Ala)(8)-NH(2), Ac-(Aib-Ala)(4)-NH(2), and Ac-(Aib)(8)-NH(2) in 3(10)- and alpha-helical conformations.     

Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy: effect of potassium ions on Nephila spidroin films

We used time-resolved Fourier transform infrared spectroscopy (FTIR) to follow a conformation transition in Nephila spidroin film from random coil and/or helical structures to beta-sheet induced by the addition of KCl from 0.01 to 1.0 mol/L in D(2)O. Time series difference spectra showed parallel increases in absorption at 1620 and 1691 cm(-)(1), indicating formation of beta-sheet, together with a coincident loss of intensity of approximately 1650 cm(-)(1), indicating decrease of random coil and/or helical structures. Increase in KCl concentration produced an increased rate of the conformation transition that may attributable to weakening of hydrogen bonds within spidroin macromolecules. The conformation transition was a biphasic process with [KCl] > or = 0.3 mol/L but monophasic with [KCl] < or = 0.1 mol/L. This suggests that, at high KCl concentrations, segments of the molecular chain are adjusted first and then the whole molecule undergoes rearrangement. We discuss the possible significance of these findings to an understanding of the way that spiders spin silk.     

Effect of P-chirality of internucleotide bonds on B-Z conversion of stereodefined self-complementary phosphorothioate oligonucleotides of the [PS]-d(CG)4 and [PS]-d(GC)4 series

Diastereomerically pure, partially modified (in selected positions) or fully modified phosphorothioate oligomers of the [PS]-d(CG)(4) and [PS]-d(GC)(4) series were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. NaCl induces the B-Z transition of [All-S(P)R(P)-PS]-d(CG)(4) with a midpoint of transition at ca. 2 M, which is approximately 1 M less than for unmodified d(CG)(4). Also, [All-R(P)S(P)-PS]-d(GC)(4) at 5 M NaCl converts to the Z form to the extent of ca. 55%, while the unmodified d(GC)(4) counterpart does not convert at all. This enhanced ability of stereodefined phosphorothioate oligomers to adopt the Z conformation is discussed in terms of already known structural factors (hydrogen bonding and water bridges) facilitating the B-Z transition, identified for unmodified d(CG)(n) oligonucleotides. By CD spectroscopy, the [All-S(P)-PS]-d(CG)(4) oligomer at a NaCl concentration higher than 0.01 M adopts a unique conformation as assessed from the presence of an additional negative band centered at 282 nm.     

Importance of coulombic end effects on cation accumulation near oligoelectrolyte B-DNA: a demonstration using 23Na NMR.

The local cation concentration at the surface of oligomeric or polymeric B-DNA is expected, on the basis of MC simulations (Olmsted, M. C., C. F. Anderson, and M. T. Record, Jr. 1989. Proc. Natl. Acad. Sci. USA. 86:7766-7770), to decrease sharply as either end of the molecule is approached. In this paper we report 23Na NMR measurements indicating the importance of this "coulombic" end effect on the average extent of association of Na+ with oligomeric duplex DNA. In solutions containing either 20-bp synthetic DNA or 160-bp mononucleosomal calf thymus DNA at phosphate monomer concentrations [P] of 4-10 mM, measurements were made over the range of ratios 1 < or = [Na]/[LP] < or = 20, corresponding to Na+ concentrations of 4-200 nM. The longitudinal 23Na NMR relaxation rates measured in these NaDNA solutions, Robs, are interpreted as population-weighted averages of contributions from "bound" (RB) and "free" (RF) 23Na relaxation rates. The observed enhancements of Robs indicate that RB significantly exceeds RF, which is approximately equal to the 23Na relaxation rate in an aqueous solution containing only NaCl. Under salt-fre-tconditions ([Na]/[P] = 1), where the enhancement in Robs is maximal, we find that Robs--RF in the solution containing 160-bp DNA is approximately 1.8 times that observed for the 20-bp DNA. For the 160-bp oligomer (which theoretical calculations predict to be effectively polyion-like), we find that a plot of Robs v. [P]/[Na] is linear, as observed previously for sonicated (approximately 700 bp) DNA samples. For the 20-bp oligonucleotide this plot exhibits a marked departure from linearity that can be fitted to a quadratic function of [P]/[Na]. Monte Carlo simulations based on a simplified model are capable of reproducing the qualitative trends in the 23Na NMR measurements analyzed here. In particular, the dependences of Robs--RF on DNA charge magnitude of Z(320 vs. 38 phosphates) and (for the 20-bp oligomer) on [Na]/[P] are well correlated with the calculated average surface concentration of Na+. Thus, effects of sodium concentration on RB appear to be of secondary importance. We conclude that 23Na NMR relaxation measurements are a sensitive probe of the effects of oligomer charge on the extent of ion accumulation near B-DNA oligonucleotides, as a function of [Na] and [P].     

Mn2+ and other transition metals at low concentration induce the right-to-left helical transformation of poly[d(G-C)].

The effects of the first-row transition metal ions on the right(B)- to left(Z)-handed helical transition of poly[d(G-C)] have been determined. The Z conformation is induced by MnCl2 at submillimolar concentrations. The forward reaction has a very large activation energy (440 kJ/mol) so that a facile conversion occurs only at temperatures above 45 degrees C. However, the left-handed form remains stable upon cooling. The addition of ethanol (20% v/v) eliminates the requirement for elevated temperature. The transition is highly co-operative and is accompanied by spectral changes (absorption, circular dichroism) characteristic for the B----Z conformational transition. NiCl2 and CoCl2 also induce the B----Z transition in poly[d(G-C)] but the activation energies and thus the temperature requirements for the forward reaction are lower than those observed with MnCl2. The left-handed DNA formed in the presence of Mn2+ is similar to 'Z DNA' previously described in Mg2+-EtOH (van de Sande and Jovin , 1982): (a) it readily sediments out of solution at low speed as a consequence of intermolecular association which, however, is not accompanied by turbidity; and (b) it supports the binding of ethidium bromide although this drug interacts preferentially with the B form of DNA. With Ni2+, the B----Z isomerization step can be separated from the subsequent specific Z----Z* association. Mn2+, Ni2+, and Co2+ also promote the B----Z transition of poly[d(G-m5C)] at substoichiometric concentrations with respect to DNA nucleotide.     

Comparison of alpha-lactalbumin and lysozyme using vibrational circular dichroism. Evidence for a difference in crystal and solution structures

The conformation of the milk protein alpha-lactalbumin has been studied using vibrational circular dichroism (VCD) and compared to parallel studies on lysozyme. These proteins have been shown by Acharya et al. [(1989) J. Mol. Biol. 208, 99-127] to have very similar three-dimensional crystal structures. However, their VCD spectra in D2O solution are quite different. The VCD of lysozyme in D2O more resembles that of alpha-lactalbumin in 33% propanol/D2O, under which conditions alpha-lactalbumin has conformationally transformed to a structure with increased helical fraction. These results can be seen to be consistent with UVCD and resolution-enhanced FTIR spectra of alpha-lactalbumin and lysozyme in both D2O and H2O environments. The solvent sensitivity of the alpha-lactalbumin spectra and hence of its conformation contrasted with the lack of such sensitivity for lysozyme suggest that the alpha-lactalbumin crystal structure represents a conformation different from that which is dominant in aqueous solution.     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr³⁺ ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr³⁺ ion binding mainly to N₇ (G) and to phosphate groups was demonstrated. ψ‐Type VCD spectra resembling electronic CD (ECD) spectra, which appear during ψ‐type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large‐scale helicity. Atomic force microscopy confirmed DNA condensation by Cr³⁺ ions and the formation of tight DNA particles responsible for the ψ‐type VCD spectra. Upon increasing the Cr³⁺ ion concentration the shape of the condensates changed from loose flower‐like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr³⁺ ions studied. The secondary structure of DNA remained in a B‐form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles. © 2002 Wiley Periodicals, Inc. Biopolymers 61: 243–260, 2002     

Conformational study of linear alternating and mixed D- and L-proline oligomers using electronic and vibrational CD and fourier transform IR

Vibrational CD (VCD) spectra of a series of blocked linear, alternating D - and L -proline containing oligopeptides, dissolved in D₂O and in CDCl₃. are reported. For the Boc-LDL -Pro₃ to Boc-DLDLDLDL-Pro₈ oligomers. The VCD spectra in the amide I band is a positive couplet, opposite in sense to that obtained for (L -Pro)_n oligomers. While this admits the possibility of their favoring a right-handed helical chain conformation, the amide I ir spectra for these dl oligomers in D₂O indicate a mixed, apparently alternate, cis-trans conformation that prevents a simple conclusion. Their VCD in D₂O evidence no narrowing and has a progressive loss in intensity (measured as Δ /A,) with an increase in chain length. In CDCl₃a similar pattern of positive VCD couplets decreasing in intensity with length was seen, but their spectra are narrower. Their electronic CD (ECD) in the uv, also indicates a loss in intensity with increasing length. Oligomers with odd or even numbers of Pro residues have different ECD patterns, indicating that those spectra are strongly influenced by local contributions arising in the N-terminal groups. The VCD arises from dipolar and vibrational coupling of the amides in the helical structure. All the spectra are consistent with the chiral end groups leading to formation of an excess of one helical handedness. With an increase in length, the influence of this selectiveness is less and the overall CD measured decreases. © 1995 John Wiley & Sons, Inc.