Circular Dichroism Pattern Research Articles

Binding-induced conformational transition of sodium poly-L-glutamate by iron(III) complex ions in aqueous solution

The binding of pseudo-octahedral trans- and cis-FeIII complex ions by sodium poly-L-glutamate (PLG) and dextransulphate (DS) in aqueous solution at about pH 7 has been studied. Equilibrium dialysis and “phase-separation” data show that the affinity for the complex counterions by both polyelectrolytes follows the order trans-[Fe(tetpy)(OH)2]+ > cis-[Fe(pmen)(OH)2]+. Evidence of a specific site binding, leading to a marked “renaturation” effect on the charged polypeptide, is produced in the case of the trans-FeIII-quaterpyridine compound. The binding isotherm of the trans-complex + PLG system and the circular dichroism patterns of the polypeptide as a function of the bound-trans-complex to polymer-residue ratio were successfully treated by a two-state model for the polyelectrolyte and a preferential association of the complex ions to the helical conformation of the polymeric matrix. All these features are examined in the light of the structural characteristics of the interacting species. The implications of the different stereochemistry of the other complex ions studied on the binding process with PLG are also discussed.

Circular Dichroism Spectra of Cobalt(III) Complexes Containing One or Two Azido, Isothiocyanato, and Nitro Ligands. I. The Bis(ethylenediamine) Complexes of cis Type

Abstract Circular dichroism spectra have been measured for the cis[Co(X)(NH3)(en)2]2+, cis-[Co(x)2(en)2]2+, and cis-[Co(X)(X′)(en)2]+ complexes in the visible and ultraviolet region (X and X′=N3−, NCS−, or NO2−). The absolute configurations of the complexes have been assigned from the circular dichroism pattern in the first d-d absorption band, region and correlated with that in the near ultraviolet region of the so-called specific absorption band. The origin of the specific absorption band has been discussed.

Synthesis and characterization of an ionizable polyhexapeptide collagen model

AbstractPoly(Lys(HBr)‐Gly‐Pro‐Pro‐Gly‐Pro) has been synthesized and studied by circular dichroism (CD) spectroscopy. It is apparently the first polyhexapeptide collagen model reported with an ionizable side chain. The monomer (ε‐(p‐nitrobenzyloxycarbonyl)‐Lys‐Gly‐Pro‐Pro‐Gly‐Pro‐p‐nitrophenyl‐ester) was prepared by a stepwise strategy employing active esters. Polymerization in N,N‐dimethyl formamide, followed by removal of the Lys side chain protection with HBr/acetic acid, gave a polydisperse product. Fractionation was accomplished by gel filtration chromatography. The polydisperse material had a molecular weight (Mr = 5–17,000). High molecular weight fractions from triple helices under concentrated conditions at 2°C. The triple helical structure gives a CD pattern very similar to that of collagen and its triple helical analogs. However, unlike collagen, the polyhexapeptide undergoes spontaneous dissociation at temperatures substantially below the melting temperature from a triple helical form to single chains. This process is promoted at low concentrations, high temperature, neutral pH, and low molecular weight, and is apparently due, in large part, to unfavorable ionic side‐chain interactions. In addition to this relatively slow “ionic” dissociation the triple helical polypeptide may be thermally dissociated in a manner similar to collagen. The thermal denaturation is a relatively fast process compared with ionic dissociation.A high molecular weight fraction (3 × Mr = 48,000) was found to melt at 42°C at neutral pH but increased to 54°C at pH 12 where the lysyl side chains are predominantly deprotonated. Furthermore, reconstitution of triple helices appeared to be more readily achieved at high pH. Thus it is concluded that ionic repulsion between side chains causes destabilization of the triple helix and hinders reconstitution.

Circular Dichroism of trans-Dichlorocobalt(III) Complexes with Chiral 1,2-Diamines

Abstract The circular dichroism (CD) spectra of trans-dichlorobis(diamine)cobalt(III) complexes were measured in methanol solution in the 700–200 nm region, where the diamines have the general formula of NH2CH(R)CH2NH2(R=isopropyl, isobutyl, sec-butyl, benzyl and phenyl) and belong to the S configuration except the phenyl-substituted one. The spectra are essentially the same as thq spectrum of the (S)-propylenediamine ((S)-pn; R= methyl) complex, except for the (R)-C-phenylethylenediamine ((R)-phenen) complex. The variation of the C-substituted alkyl groups has hardly any influence on the CD of the trans-[CoCl2(diamine)2]+ type complexes, demonstrating that the vicinal contribution from the C-substituents can be disregarded in these compounds. The trans-[CoCl2((R)-phenen)2]+ ion shows a CD pattern differing significantly from that of the trans-[CoCl2((R)-pn)2]+ ion in the first absorption band region, suggesting the significance of the vicinal effect due to the C-substituted phenyl group. The CD curve of trans-[CoCl2((−)D-stien)2]+ ion (stien=stilbenediamine) was found to be antimeric to that of the (R)-phenen complex. Taking into account the anomalous vicinal effect of the phenyl groups, the absolute configuration of the (−)D-stien was assigned to the (S,S) configuration.

Circular dichroism and absorption of the polytetrapeptide of elastin: A polymer model for the β-turn

Circular dichroism and absorption data are reported for the polytetrapeptide of elastin, HCO-(Val-Pro-Gly-Gly)40-Val-OMe. The circular dichroism pattern, with a negative band at 224 nm, a positive band at 205 nm and a second negative band below 190 nm, is distinct from the circular dichroism patterns exhibited by the α-helix, β-pleated sheet and random coil conformations. As previous nuclear magnetic resonance studies have demonstrated that the polytetrapeptide is comprised of repeating β-turns, the absorption and circular dichroism data is presented as a model system for the β-turn conformational feature which is commonly found in globular proteins. The absorption and circular dichroism data are resolved into component Gaussian curves.

Effect of pH on the Circular Dichroism(CD)-sensitive Transitions of Some Lanthanoid(III) Complexes and “Pseudo CD-sensitive Transitions” of Europium(III) and Terbium(III) Complexes

Abstract The effect of pH on the CD-sensitive transitions of lanthanoid(III) complexes with l-propylenediaminetetraacetic acid(l-PDTA) and l-ethylenediaminedisuccinic acid(l-EDDS) was studied. The relation between the cross-over pH where the CD pattern changes or sign of CD reverses and the number of 4f-electrons in lanthanoid(III) complexes with l-EDDS showed a tetrad curve. A strong evidence for coordination of hydroxide ions was suggested. “Pseudo CD-sensitive bands” in the spectra of the complexes of europium(III) and terbium(III) which do not obey the selection rule ΔJ≤1 were found, and the mechanisms of these transitions were elucidated.

Isolation of synaptic complexes in a CsC1 gradient: conditions for maximal resolution in the zonal rotor B-XIV and circular dichroism patterns.

Abstract Synaptic complexes were isolated from different brain regions and developmental stages in a CsCl density gradient using a Ti-14 zonal rotor. The buoyant density of the synaptic complexes from all these tissues was 1.178–1.190. The conditions for maximal resolution were rapid displacement of the density gradient from the rotor (40 ml/min); continuous centrifugation of the particles in the gradient for 66 hours, sample loads not exceeding 200 mg membrane protein. The banding densities of the membranes in the CsCl gradient were shown to be a linear inverse function of their lipid content. The circular dichroism patterns of synaptic complexes and other neural membranes in suspension or SDS solutions were similar to those of membranes from other mammalian cells or from bacteria although the ellipticities of the neural membranes were lower. These studies indicate that the proteins in a variety of membranes are in an α-helical conformation.

RNA polymerase-DNA complexes I. The study of the conformation of nucleic acids at the growing point of RNA in an RNA polymerase-DNA system

Complexes of RNA polymerase with the DNA regions protected against deoxyribonuclease digestion by the enzyme molecule (“kegs”) were obtained and studied by the method of circular dichroism (CD). The DNA within the keg consists of 20–25 base pairs and has the CD spectrum of B-form DNA. An A-like CD pattern, however, appears when the kegs start to synthesize RNA. This may be due to: (1) CD of the growing RNA chain (if it is stacked), (2) a hybrid of RNA · DNA (which is known to be in the A-form), or (3) a conformational B → A transition of the DNA template. However, the CD magnitude for the synthesizing kegs is too great for Explanation 1 to be likely.

Separation of contributions of tryptophans and tyrosines to the ultraviolet circular dichroism spectrum of hen egg-white lysozyme.

Ultraviolet circular dichroism measurements over the wavelength range of 190–300 nm have been carried out on hen egg‐white lysozyme and on an iodine‐oxidized derivative of lysozyme in which Tryptophan‐108 has been selectively modified. The contribution of Tryptophan‐108 to the aromatic circular dichroism profile has been ascertained from a difference spectrum generated between the native and the modified protein. This tryptophyl residue is exceptional in its properties, as it is the principal contributor to the circular dichroism pattern in this wavelength range. The chemically modified protein shows only minor differences from the native molecule in terms of the magnitude of the conformation‐dependent ellipticity bands at 209 and 192 nm. Separation of the partial contributions of tyrosines was obtained from circular dichroism difference spectra constructed from circular dichroism profiles of both the native and iodine oxidized proteins at pH 7 and 11.2. Oligomers of N‐acetyl‐d‐glucosamine, which are inhibitors of lysozyme, markedly affect the circular dichroism spectrum of the enzyme at the aromatic region. It is postulated that this effect on the spectrum is a result of a change in orientation of Tryptophan‐108, which occurs on binding of the inhibitors to the active site of the enzyme.These results are discussed with respect to the proposed three‐dimensional model of lysozyme.

Conformation of cytochromes: IV. Conformational homology of functionally homologous cytochromes c

The conformational homology of functionally homologous cytochromes c from horse, beef, tuna, turkey, pigeon and moth has been investigated by comparison of the circular dichroism (CD) spectra of each preparation in both oxidation states of iron and in the wavelength region 600-185 mμ at pH 6.8. In the oxidized state all preparations exhibit similar dichroic features with only small variations in the relative magnitudes of various extrema, except for the aromatic circular dichroism curve for tuna cytochrome c. The preparations in the reduced state of iron also exhibit comparable dichroic spectra, both in the region of the Soret transition and the intrinsic absorption region. The CD patterns for the reduced cytochromes in the α-β absorption region, however, exhibit deviations suggestive of small but distinct differences in the electronic asymmetry of the prosthetic groups, especially of the porphyrin moiety, possibly because of differences in the electronically coupled chromophores. A gross conformational homology among various mammalian-type cytochromes c has, therefore, been suggested. The dichroic differences in the case of tuna cytochrome c have been imputed to be a reflection of an unusually asymmetric environment of the residue at position 33 of the polypeptide chain. The intrinsic CD curves above 200 mμ for all the preparations in both valence states of heme iron exhibit features characteristic of the presence of α-helical structures in the molecule. Below 200 mμ, however, the dichroic curves exhibit a single positive maximum at about 194 mμ and a definite minimum at 190 mμ. The former feature is usually associated with systems containing β-structure. The presence of a minimum at about 190 mμ, when taken in conjunction with the characterizing features for α-helical models (a large positive maximum at this wavelength), indicates the absence of α-helically organized structure in the molecule. An analysis of the conformation of the protein moiety in terms of proportions of reference model conformations (α-helix, β-structure and random coil) and based on the recently developed isodichroic method 24 confirms the low α-helical content of various cytochromes and further demonstrates the gross polypeptide conformational homology for various functionally homologous preparations.

Differential scatter of left and right circularly polarized light by optically active particulate systems.

Because of the many heterogeneous systems which are of interest to the chemist and biochemist, the problems of distortions in circular dichroism patterns have been investigated. Specifically in this communication it is shown with a relatively well-characterized particular system (a suspension of alpha-helical poly-L-glutamic acid) that there is a measurable differential scatter of left and right circularly polarized light by suspensions of the optically active particles. This is a specific example of Perrin's assertion in 1942 (Perrin, F., J. Chem. Phys., 10, 415 (1942)) that the polarization characteristics of scattered light would differ depending on whether or not the scattering particle was optically active. Differential scatter is included with the concentration obscuring effects to demonstrate that distorted circular dichroism spectra on poly-L-glutamic acid suspension can be calculated with satisfying accuracy. The approach should be applicable to correcting the circular dichroism spectra for the many particles of biological interest, e.g., membranes, viruses, mitochondria, and insoluble proteins and polypeptides, and for small crystals in an effort to answer the crystal solution problem.

The Structure and Enzyme-Coenzyme Relationship of Supernatant Aspartate Transaminase after Dye Sensitized Photooxidation

Abstract Photooxidation of supernatant glutamate aspartate transaminase, limited to the stage during which much of the enzyme activity and only histidine residues are destroyed, does not induce gross protein structural changes as judged by ultracentrifugation, optical rotatory dispersion in the ultraviolet region, and microcomplement fixation techniques. Photooxidized apoenzyme has been shown to bind at least 90% as much coenzyme as the native enzyme and forms holopyridoxal or pyridoxamine enzyme which is very similar to the native enzyme in optical and absorption spectral properties in the visible region of the spectrum. The absorption spectrum of photooxidized apo- or holoenzyme has a new maximum at 325 mµ. Because of analogy to the absorption of the photooxidation product of a model compound, glycyl-histidyl-glycine, the new maximum in photooxidized transaminase has been assigned to the photoproduct of the altered histidines in the enzyme. Circular dichroism measurements of apoenzyme revealed positive ellipticity bands in the 280 to 300 mµ regions of the spectrum. They remain essentially unaltered after photooxidation. Binding of pyridoxal phosphate to this apoenzyme results in the appearance of the well known positive ellipticity bands in the visible region and formation of new negative ellipticity bands centered at 298 and 290 mµ in the aromatic region. Photooxidation eliminates the 290 mµ band from the pyridoxal form of the holoenzyme. The circular dichroism pattern of pyridoxamine phosphate bound to photooxidized transaminase is identical with that of native pyridoxamine holoenzyme only if this enzyme form is produced by transamination of photooxidized pyridoxal holoenzyme, not if formed by addition of pyridoxamine phosphate to photooxidized apoenzyme. Another physical anomaly resulting from photooxidation is the shift of the bound pyridoxal phosphate pK from 6.3 to 6.8. Since controlled photooxidation results in modification of only histidyl residues, this shift in pK may be due to the modification of a histidine in the steric vicinity of the pyridoxal phosphate at the active site, resulting in a localized perturbation of the environment of the coenzyme. The role of the histidyl residue at the active site cannot be associated with coenzyme binding.

Coacervation of solubilized elastin effects a notable conformational change.

WHEN fibrous elastin is rendered water soluble by partial hydrolysis the product, α-elastin, undergoes a temperature elicited coacervation which is readily reversible1,2. Below room temperature α-elastin is soluble in dilute aqueous buffers in the region of its isoelectric point. On raising the temperature to 37° C a phase separation or coacervation occurs. In this communication we wish briefly to report that the circular dichroism pattern of soluble elastin most nearly resembles that of disordered proteins and polypeptides whereas the circular dichroism pattern of the coacervate (a mucilaginous precipitate) is characteristic of those obtained for the highly helical proteins and polypeptides. A calculation based on model polypeptides and myoglobin and on corrections for dampening of CD patterns due to absorption flattening and dispersion distortions3 implies an approximate 50 per cent α-helical content for the protein in the coacervate.

Correlation of light scattering and absorption flattening effects with distortions in the circular dichroism patterns of mitochondrial membrane fragments

The circular dichroism and optical rotatory dispersion patterns of biological membranes resemble, in gross aspect, those of the α-helix but the extrema are red shifted and of low amplitude. Such patterns are not typical for solublized membrane lipoproteins but rather are exhibited by the aggregates of membrane proteins (Steim and Fleishcher, 1968), as well as by aggregated polypeptides and antibiotics ( Urry and Ji, 1968 b). Since particulate systems exhibit flattened absorption bands ( Duysens, 1956) and light scattering, it is of interest to experimentally relate changes in the circular dichroism patterns with these effects. Absorption flattening results in a decrease of absorbance due to a decrease in the effective concentration of absorbers, while light scattering results in a light intensity loss and thereby an increase in absorbance as measured by the phototube. Light scattering also results in a concentration obscuring effect. The extent of light scattering and absorption flattening can differ slightly for the left-handed and right-handed circularly polarized light. Thus these effects could accentuate distortions in the ORD and CD patterns of particulate systems as compared to the molecularly dispersed samples. Previously it has been demonstrated that the low amplitude of the membrane type CD curve can be generated from the characteristic α-helix CD pattern by applying correction factors for light scattering and absorption flattening ( Urry and Ji, 1968 a & b). In this communication it is shown that the red shift and decrease in amplitude of the CD extrema of particulate systems correlate with changes in the intensity of scattered light and with the extent of absorption flattening as measured, respectively, at wavelengths where significant absorption does not occur and at wavelengths corresponding to major absorption bands.

Distortions in circular dichroism patterns of particulate (or membranous) systems

Two sources are proposed for the distortion of circular dichroism patterns of particulate systems. One is the absorption flattening of Duysens and the second, for which equations are derived, is a dispersion distortion due to light scattering. Both result in wavelength-dependent decreases in the number of absorbing units. As such the Kronig-Kramers transforms which relate circular dichroism and optical rotatory dispersion are valid and shifts and decreases in magnitude observed with one physical measurement will be reflected in the other. The Duysens absorption flattening results in maximum damping at the wavelength of the absorption peak. The dispersion distortion results in maximum damping at positions defined by the half-width of the absorption band in accord with anomalous dispersion in absorption regions. In terms of polypeptide circular dichroism this results in greatest damping of ellipticity near 208 mμ. When these wavelength-dependent effects are applied to the circular dichroism pattern of helical poly-γ-methyl- l-glutamate the circular dichroism curve characteristic of membranes is obtained. Thus the red shift and decreased magnitude of membrane CD curves are in part the result of the particulate nature of a system wherein about half of the polypeptide exhibits the features of an α-helix.

Magnetooptical Experiments on Broad Absorption Bands in Solids

The F center is discussed as an example of a point defect in solids with an associated broad absorption band. It is shown how the effect of a magnetic field on such a center can be studied by means of the Faraday effect with reference to the method of moments. The appropriate dispersion relation for obtaining the circular dichroism pattern from the Faraday rotation pattern is derived. The procedure used to evaluate numerically the dispersion relation is briefly described, and results for the F center in KI are compared with a direct observation of the effect of a magnetic field on the F band published elsewhere.