Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

Total optical activity of agarose: relation to observable transitions in the vacuum ultraviolet

The changes in optical activity that accompany and characterize the coil-helix and helix-coil transitions of agarose in aqueous solutions and gels have been investigated by combined quantitative analysis of data from vacuum ultraviolet circular dichroism (VUCD) and optical rotary dispersion (ORD). VUCD of agarose in the high-temperature coil state shows a single accessible Gaussian band centered at ～183 nm. In the helix state this band is blue-shifted by ～9 nm, and the intensity is increased by a factor of ～2.6. Spectra at intermediate temperatures can be fitted to within experimental error by linear combination of coil and helix spectra, the relative proportions required providing an index of the extent of conformational ordering. ORD spectra throughout the conformational transition have a common form and differ only in absolute magnitude. The temperature course of conformational ordering derived from ORD intensity is in close agreement with the values obtained from VUCD. In both the coil and helix states the accessible VUCD band is positive, while the overall ORD is negative, indicating strong negative CD activity at lower wavelength. The ORD contribution corresponding to the positive VUCD band was calculated by Kronig–Kramers transform, and it was subtracted from the total ORD to give the residual ORD from all other optically active transitions of the molecule. In both the coil and helix states, this residual ORD could be fitted to within experimental error by a single Gaussian CD band at ～149 nm. A negative band at this wavelength has been reported previously for agarose films, but the observed intensity, relative to that of the lower energy positive band, is substantially smaller than the fitted value under hydrated conditions. In both the coil and helix states the total optical activity of agarose, characterized by observed ORD spectra, can be matched to within experimental error by Kronig-Kramers transform of the 149-nm negative band and the smaller positive band at higher wavelength, with no necessary involvement of deeper-lying transitions. The significance of this conclusion for fundamental understanding of carbohydrate optical activity is discussed.     

Circular dichroism studies of copper(II)- hyaluronic acid complexes in relation to conformation of the polymer

The study of the Cu(II)-hyaluronate complexes by absorption and CD spectra, as well as by acid–base titration and viscosity, provides information about the nature of ligands and the conformation of the polymer. Three different complexes have been identified. The first (complex I), which is formed between pH 3 and 6, involves mainly the carboxyl groups of the polymer as ligands and is characterized by a strong absorption band at 238 nm. In this complex formation, the CD properties of hyaluronate do not charge appreciably. The second (complex II) forms between pH 6 and 8 bad shows a major change in CD properties. The changes include (1) a new positive CD band at 250 nm and a strong negative on in the π → π* amide transition region and (2) the disappearance of the negative n → π* amide CD band near 210 nm. A sharp increase in absorbance at 238 nm from complex I to II has been attributed to a conformational transition which is also manifested in the CD features of hyaluronate. Complex II involves, in addition to the carboxyl group, the nitrogen atom of the deprotonated acetamido group coordinated to Cu(II). The absorption at 230–280 nm is associated with the optically active charge-transfer transitions involving ligands to metal ion. At higher concentrations of the polymer or at higher pH, complex II aggregates to a gel, complex III. Chondroitin, differing from hyaluronic acid in the C-4 hydroxyl group configuration of the glucosamine moiety, does not show any CD change in the presence of Cu(II).The results provide further support to our fourfold helical structure of Cu(II)–hyaluronate complex at pH between 6 and 8. Intrinsic viscosities of hyaluronate in the presence of the cupric ion is lower than in the presence of other monovalent or bivalent cations, indicating a compact conformation of the polymer when it is complexed with Cu(II).     

Circular dichroism and conformational properties of modeccin,a toxic protein

According to its circular dichroism (CD) spectrum, modeccin, a toxic lectin from the roots of the South African plantModecca digitata, is structurally similar to the ricins and abrins. In nearly neutral and weakly alkaline solutions (pH 7.6–9.0) the CD spectra of modeccin displayed a positive CD band at 190–195 nm and a negative band at 210–220 nm, indicating the presence of some α-helix and β-sheet structures. In the near-ultraviolet zone, we observed positive CD bands at 232 and 245 nm and weak negative bands at 285 and 293 nm. In more strongly alkaline solutions of pH 9.5–10.2 the CD bands in the farultraviolet zone were not affected, but the CD band at 232 nm diminished and the CD band at 245 nm was enhanced. These transitions were reversible. At pH 11.2–11.5 the CD band at 232 nm disappeared completely, and the CD bands in the far-ultraviolet diminished. The CD bands at 285 and 293 nm were affected very little by the alkali, and these bands were assigned to buried tryptophan side chains. Sodium dodecyl sulfate and 2,2,2-trifluoroethanol disorganized the tertiary structure of modeccin and reconstructed the secondary structure into a new form with a higher helix content than in the native protein.     

Optical activity of saccharides—the vacuum-uv origin of sodium-D rotation

A semiempirical theory of saccharide optical activity indicates that the dominant source of Na_D rotation is a vacuum-uv CD band near 150 nm, a band observed experimentally in polysaccharide film CD spectra. The model is a modification of polarizability theory in which high-energy electronic excitations are coupled by degenerate perturbation theory, giving rise to “molecular excitons.” The existence of an excitation mode well separated in energy from even higher energy modes arises from the local symmetry of tetrahedral carbon atoms in a puckered ring structure. Calculated Na_D rotations correlate well with experimental values.     

Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process.

In the bleaching process of cephalopod rhodopsin, a new intermediate was found in the conversion process from lumirhodopsin to metarhodopsin. This intermediate of octopus has an absorption peak at about 475 nm and has been named as M475. The circular dichroism value of M475 is too small to be evaluated. On the other hand, lumirhodopsin shows a negative CD at 470 nm, a positive CD at 350 nm and a large positive CD band with three peaks at 280, 287 and 295 nm. Such a large CD band in the ultraviolet region is not observed in rhodopsin, M475 and metarhodopsin. This CD seems to be mainly due to tryptophan and tyrosine residues restricted in free rotation in the protein moiety of lumirhodopsin. The intermediate in the photoregeneration process of cephalopod rhodopsin, P380, has a positive CD band at the main peak, 380 nm, and also a large positive CD band in the ultraviolet region like lumirhodopsin.     

Temperature dependence of the optical activity of human serum low density lipoprotein. The role of lipids.

Low density lipoprotein (LDL) (1.024-1.045 G/cm3) was prepared by ultracentrifugal flotation from serum of normal fasting subjects. Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra in the ultraviolet region were measured at 2, 25, and 37 degrees on LDL, lipid extracted from LDL, and on pure component lipids. All exhibit reversible, temperature-dependent optical activities. Sphingomyelin has a strong negative CD band around 195 nm. Cholesterol and cholesteryl esters have a CD minimum at 208 nm. They have positive CD bands around 201 and 198 nm which decrease sharply and become negative at 198 and 193 nm, respectively. The CD of the total lipid extract of LDL is negative and drops monotonically below 200 nm. Thus, the lipid moiety could account for the increasing negativity of the CD of LDL below 195 nm. After subtraction of the ellipticity corresponding to amounts of lipids in organic solvents equivalent to those found in LDL, the 208-210 nm trough of LDL diminishes markedly. This is accompanied by a blue-shift of the extrema from 195-196 to 193 nm and an increase in the magnitude of the positive ellipticity. The fractions of helix and of beta form in the protein, determined by the method of Y. H. Chen, J. T. Yang, and K. H. Chau ((1974), Biochemistry 13, 3350), in the wavelength interval of 250-240 nm, remain essentially unchanged between 2 and 37 degrees. These observations suggest that a substantial part of the thermal change in the CD spectrum of LDL between 208 and 210 nm may be attributable to lipids.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.     

Optical activity of octopus metarhodopsins.

The optical activity of octopus rhodopsin, acid metarhodopsin and alkaline metarhodopsin was studied by a sensitive and rapid CD apparatus. For sometime it has been thought that cephalopod metarhodopsins do not have any optical activity associated with their main absorption band. However, the present work shows that acid metarhodopsin in digitonin has a positive CD band at 498 nm and a negative CD band at 436 nm and alkaline metarhodopsin has a negative CD band at 381 nm. Detergent affected the wavelength of the CD peak of the visual pigments though the pattern of the spectrum was similar. From these results it is concluded that the conformation of all-trans retinal in octopus metarhodopsin is influenced by the asymmetric conformation of the protein near the retinal and therefore inducing optical activity.     

Circular dichroism of lipoxygenase-1 from soybeans.

The circular dichroism spectra of the three forms of lipoxygenase-1 from soybeans show characteristic differences in the region between 300 and 600 nm. Native lipoxygenase-1 only shows a negative dichroic band around 330 nm. Yellow lipoxygenase-1, obtained by addition of an equimolar amount of 13-F-hydroperoxylinoleic acid to the native enzyme, shows a positive Cotton effect at 425 nm, while the negative band band at 330 nm has increased in intensity. The blue enzyme, representing a complex of yellow enzyme with 13-L-hydroperoxylinoleic acid exhibits a negative dichroic band at 580 nm and positive bands at 410 and 391 nm. The near-ultraviolet CD spectra of the three forms of lipoxygenase are very similar, showing several well resolved positive dichroic bands at 0 degrees C. Using the method of Chen et al. (Chen, Y.-H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131) the contents of alpha-helix, beta- and unordered form of native lipoxygenase-1 were estimated to be 34, 27 and 39% respectively.     

Vacuum-ultraviolet circular dichroism of sodium hyaluronate oligosaccharides and polymer segments

The CD spectrum of an enzymatically derived sodium hyaluronate (NaHA) segment preparation with chain length 18 ± 3 disaccharide units [NaHAseg, ( NaGlcUA GlcNAc)_15–20°. NaGlcUA, sodium D -glucuronate; GlcNAc, 2-acetamido-2-deoxy-D -glucose] in H₂O was recorded to 180 nm using a computer-controlled vacuum-uv CD instrument. Near 190 nm the spectrum is of low intensity, similar to the sum of the free monosaccharide contributios, attributed to the π–π* transitions of the acetamido and carboxylate substituents. In contrast, much smaller oligosaccharides, also derived from high-molecular-weight NaHA by enzymatic digestions, show CD spectra in H₂O with prominent bands centered near 190 nm. The oligosaccharide spectra can be matched as linear combinations of interior sugar residue (? NaHAseg) and end sugar residue CD contributions. End residues from oligosaccharides of the type (NaGlcUA-GlcNAc)_n show a negative CD band near 190 nm. End residues from oligosaccharides of the reverse sequence (GlcNAc-NaGlcUA)_n show a positive CD band near 190 nm. Averaging of the two end-residue spectral contributions yields an approximate match for the spectrum of NAHAseg below 200 nm. It is proposed that the low intensity CD of NaHA in the π–π* region is the result of large-magnitude, oppositely signed contributions, which can be visulized by studying oligosaccharides.     

Conformation of ribooligonucleotide duplexes containing an alternating C-G sequence which show an unusual circular dichroism spectrum

The poly[r(G-C)] duplex shows an unusually large negative band in the long wavelength region of the CD spectrum. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized chemically and their properties were examined by UV and CD, and 1H and 31P NMR spectroscopy. These ribooligomers form a self-complementary duplex at low temperature, the CD spectrum of which shows a negative band at around 290 nm and a positive band at around 265 nm with almost equal magnitudes. The proton resonances in the 1H NMR spectra of the oligo[r(C-G)] duplexes were assigned by nuclear Overhauser effect experiments. The chemical shift-temperature profiles of the base proton signals and the sharp singlets observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. Moreover, a 500-MHz two-dimensional nuclear Overhauser effect experiment recorded for r(C-G-C-G-C-G) shows that all guanine bases adopt the normal anti-conformation. CD-temperature profiles and 31P NMR spectra of oligo[r(C-G)]s support this conclusion. These results indicate that duplexes of oligo- and polyribonucleotides containing alternating C-G sequences can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.     

Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane

We examined the effects of volatile anesthetics on the structure of the bacteriorhodopsin in the purple membrane by measurements of the absorption spectrum and the visible circular dichroism (CD) spectrum and assay of the retinal composition. As the concentrations of halothane, enflurane and methoxyflurane were increased, the absorption at 560 nm decreased but that at 480 nm increased with an isosbestic point around 510 nm. These anesthetic-induced spectroscopic changes were reversible. The CD spectrum showed the biphasic pattern with a positive and a negative band. As the concentration of halothane was increased from 4 mM to 8mM, the negative band reversibly diminished more drastically than the positive band, and at 8 mM of halothane the positive band shifted to around 480 nm. These results show that halothane disturbed the exciton coupling among bacteriorhodopsin molecules. The retinal isomer composition was analyzed using high performance liquid chromatography. The ratio of 13-cis- to all-trans-retinal was 47:53, 34:66 and 19:81 at control, 7.4 mM and 14.9 mM enflurane, respectively. After elimination of enflurane, the ratio returned to the control value. These findings indicate that volatile anesthetic directly affect a bacteriorhodopsin in the purple membrane and induce conformational changes in it.     

Circular dichroism of helical polynucleotides calculated by the linear response method

Linear response theory in the decorrelation or random-phase approximation is used to calculate the absorption and CD spectra of model helical polymers, including single-stranded polyadenylic acid. The method, which makes use of infinite polymer selection rules for the linear response tensor, has the advantages that (1) only a few three-dimensional matrices need be inverted; (2) spectral band shapes of the polymer arise naturally from those of the monomer, as well as from the geometry-dependent interactions in the helix; and (3) the spectral dependence on geometrical factors of the helix is made transparent. It is found that the structure of the polymer CD spectrum depends critically on monomer bandshape. An asymmetric CD spectrum, similar to some experimental spectra, arises from either a Gaussian or a composite monomer band. Single-stranded polyadenylic acid spectra are sensitive to helix geometry in the region 200–240 nm, in reasonable agreement with experimental spectra. This sensitivity arises from the 207-nm monomer transition, and the results suggest that this region of the spectrum should be more fully exploited as a tool for helix geometry studies.     

R-phycoerythrins having two conformations for the same aggregate

The visible circular dichroism (CD) spectrum of an R-phycoerythrin (Porphyra tenera) is composed of several positive bands. The protein in aqueous buffer very slowly exhibits changes in the CD spectrum of its chromophores, a band at 489 nm undergoes an increase in intensity and a red shift. When the band reached a 493 nm maximum, the spectrum became very stable. The aggregation state of the protein did not change during this spectral conversion. The chromophore CD spectrum was also obtained in the presence of a low concentration of urea or sodium thiocyanate, and the identical change in the CD was noted, but the change was much faster. The visible absorption and CD in the far UV spectra were unaffected by urea. Unchanged visible absorption and protein secondary structure (61% alpha helix) contradicted by comparatively salient alterations in the visible CD spectra suggested very subtle structural changes are influencing some of the chromophores. For a second R-phycoerythrin (Gastroclonium coulteri), the CD of the chromophores had a negative band on the blue edge of the spectrum. This is the first negative CD band observed for any R-phycoerythrin. Treatment of this protein with low concentrations of urea produced a change in the visible CD with the negative band being completely converted to a positive band. Fluorescence studies showed that the treatment by urea did not affect energy migration. Deconvolution of the CD spectra were used to monitor the chromophores. The results demonstrated that the same aggregate of each R-phycoerythrin could exist in two conformations, and this is a novel finding for any red algal or cyanobacterial biliprotein. The two forms of each protein would differ in tertiary structure, but retain the same secondary structures.     

Conformational changes of type II regulatory subunit of cAMP-dependent protein kinase on cAMP binding

The effect of cAMP on the conformation of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from bovine heart was investigated by UV-difference and circular dichroism (CD) spectroscopy. The UV-difference spectrum of RII with and without cAMP showed a positive band around 278 nm and a negative band at 256 nm. Similarly, cAMP enhanced the ellipticity of RII in the region between 280 and 300 nm and decreased that between 250 and 280 nm. In addition, cAMP transformed the far-UV CD spectrum of RII from that of a negative band minimally at 209 nm with a shoulder at 223 nm to one with two minima at 222 and 211 nm. These data show that cAMP induces conformational changes of RII upon binding. Such structural changes may be the basis of activation of cAMP-dependent protein kinases by cAMP.     

Synthesis and conformational studies of ribooligonucleotides which contain an alternating C-G sequence and show unusual circular dichroism spectra

The poly [r(C-G)] duplex shows an unusually large negative CD band in the long wavelength region. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized by a phosphotriester method and their properties were examined by UV, CD, 1H and 31P NMR spectroscopy. These ribooligomers form self-duplexes at low temperature, the CD spectra of which show negative bands at around 290 nm and positive bands at around 265 nm. The results of 1H nuclear Overhauser effect experiments, 1H chemical shift-temperature profiles of base protons, and the sharp singlet observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. The CD-temperature profiles and 31P NMR spectra support this conclusion. These results indicate that RNA duplexes with an alternating C-G sequence can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.     

A study of the interaction between D-amino acid oxidase and quasi-substrates.

To study the interaction between D-amino acid oxidase [EC 1.4.3.3] and quasi-substrates such as benzoate and o-, m-, and p-aminobenzoate, visible circular dichroism spectra (CD spectra) were measured and the binding rate and affinity of o-aminobenzoate to the enzyme were observed by following the absorption changes at various wavelengths. We found a new CD band around 560 nm, corresponding to the charge-transfer complexes which result from the formation of aminobenzoate complexes with the enzyme. The ellipticity of this band was positive for the p-aminobenzoate complex, but negative for the o- and m-aminobenzoate complexes. Crossover points in CD spectra were observed at 470 nm for the m-aminobenzoate complex and at 475 nm for the o-aminobenzoate complex. They probably resulted from overlapping of the positive CD band of FAD bound with the enzyme and the negative CD band of the charge-transfer complex. We propose that the amino group in aminobenzoate, not the pi-electrons of the benzene ring, is the electron donor in the charge-transfer complex and that the position of the amino group is very important for the charge-transfer interaction. The binding rate and affinity of o-aminobenzoate to the enzyme were determined using the absorption changes at 370 nm (380 nm), caused by the modification of electronic states of FAD bound with the enzyme, and at 550 nm (565 nm), caused by the formation of the charge-transfer complex of o-aminobenzoate with the enzyme. No differences between these parameters with wavelength were observed. This independence of wavelength simplifies discussion of the experimental data obtained from absorption changes.     

Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.