Circular dichroic studies of the DNA and RNA of nucleoli

Circular dichroism (CD) in the 240-300-nm region was used to study the conformation of DNA and RNA complexed with proteins in isolated nucleoli form HeLa cells. Deoxyribonuclease or ribonuclease digestion was employed to obtain (1) the individual CD spectra of nucleolar DNA or RNA in complex form with proteins, or in free form; and (2) the experimental CD baseline correction to exclude contributions from nonnucleic acid sources such as light scattering artifacts and proteins. The CD spectrum of nucleolar DNA in DNA-protein complexes was highly reduced in ellipticity in comparison with protein-free DNA. It showed a positive peak at 283 nm with a molar ellipticity [theta]283 = 1200 deg cm2 dmol-1 and a crossover at 262 nm. Addition of sodium dodecylsulfate shifted the peak to 276 nm with [theta]276 8000 deg cm2 dmol-1 and a crossover at 254 nm. The CD spectrum of nucleolar RNA in RNA-protein complexes was also reduced in comparison with protein-free RNA, showing a peak at 269 nm ([theta]269 = 6900 deg cm2 dmol-1), and a crossover at 250 nm. Addition of sodium dodecyl sulfate shifted the peak to 265 nm with [theta]265 = 18 000 deg cm2 dmol-1 and a crossover at 246 nm. The low ellipticity of both nucleolar DNA and RNA when complexed with proteins was increased by treatment with sodium chloride, urea, or heparin. This suggests that some ionic, hydrophobic, and hydrogen bondings are involved in the nucleic acid-protein interaction in nucleolar chromatin similar to that observed in nuclear chromatin.     

Thermal denaturation of sheared and unsheared chromatin by absorption and circular dichroism measurements

Thermal denaturation of chromatin is observed by simultaneously monitoring absorption and circular dichroism at 276 nm as functions of temperature. Either observation indicates that sheared chromatins shows less thermal stability than native chromatin. The temperature-dependent ellipticities at 276 nm of these chromatins show features not seen in the absorption curves: the ellipticity of unsheared chromatin increases with temperature, while this increase is abolished or greatly reduced in the same chromatin after shearing. After its first thermal transition (prior to the helix-coli transition) the unsheared chromatin achieves the same ellipticity as sheared chromatin.     

Conformational states of chromatin nu bodies induced by urea

Monomer chromatin nu bodies (nu1) from chicken erythrocyte nuclei were exposed to 0-10 M urea plus 0.2 mM EDTA (PH 7). Alterations in nu1 conformation were examined using hydrodynamic methods (i.e., S, eta, and (formula: see text)), thermal denaturation, circular dichroism, reactivity of histone thiol groups to N-ethyl maleimide, and electron microscopy. The two domains of a nu body (i.e., the DNA-rich shell and the protein-rich core) aeared to respond differently to the destabilizing effects of increasing urea; DNA conformation and stability exhibited noncooperative changes; the core protein structure revealed cooperative destabilization between 4 and 7 M urea. Companion studies on the conformation of the inner histone "heterotypic tetramer" also revealed cooperative destabilization with increasing urea concentration.     

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

Denaturing behavior of glutathione reductase from cyanobacterium Spirulina maxima in guanidine hydrochloride

The influence of guanidine hydrochloride (Gdn-HCl) on glutathione reductase from Spirulina maxima has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, thiol groups accessibility, and gel filtration chromatography. It was found that the denaturation process involves several intermediate states. At low, Gdn-HCl concentrations (Cm = 0.4 M), reductase activity was fully lost. However, below 3 M Gdn-HCl, this inhibition was freely reversible upon removal of the denaturing agent. Gel filtration experiments revealed that this reversible inhibition was not due to dissociation of the tetrameric enzyme. Structural studies strongly suggest that the conformation of this intermediate state is similar to that of native enzyme. A model in which a local region of the polypeptide chain assumes an extended conformation (D. T. Haynie, and E. Freire, Proteins 16,115-140) is proposed for the reversibly inactivated enzyme. Between 3 and 4 M Gdn-HCl (Cm = 3.5), the enzyme activity was irreversibly lost, this inhibition being concomitant with the loss of ellipticity, changes in both wavelength and intensity at the maximum of fluorescence emission, and dissociation of the enzyme into unfolded monomers; these results reveal that gross changes in the protein conformation occur under these conditions. At 4 M Gdn-HCl an equilibrium exists between the denatured forms of dimer and monomer, which is completely shifted toward the unfolded monomers at 5 M Gdn-HCl. Irreversibility in the Gdn-HCl-induced denaturation of S. maxima glutathione reductase was not due to aggregation of the unfolded enzyme.     

Assembly of DNA with histones and nonhistone chromosomal proteins in vitro

We have examined, by protein binding assays, thermal denaturation, and circular dichroism, the possible effects of histones on nonhistone chromosomal protein (NHCP) interactions with DNA. For these studies, we have fractionated mouse Krebs II chromosomal proteins into three discrete fractions: Mo, 5 M urea-soluble NHCP; M1, 5 M urea-1 M NaCl-soluble NHCP from 5 M urea-extracted chromatin; and M3, 5 M urea-3 M NaCl-soluble chromosomal proteins from 5 M urea-1 M NaCl-extracted chromatin. These fractions contain heterogeneous populations of NHCP, and were found to differentially affect histone binding to DNA by methods of reconstitution, or by direct binding of M0, M1, or M3 to urea-salt reconstituted DNA with histones. M0 was found to exert a unique effect on the thermal denaturation and circular dichroic spectra of DNA-histone complexes. M0 from Krebs II chromatin was also found to complete for DNA sites in the presence of M0 from mouse liver chromatin. In addition, in 5 M urea, pH 8.0, histone binding to DNA reached saturation at 1.85 mg/mg of DNA, higher than the in vivo ratio of 1.00 mg/mg of DNA. Saturation of histone binding to DNA occurred only in the presence of 5 M urea, resulting in a reduction of nonspecific histone-histone interactions on DNA.     

Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule

The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.     

Investigation of genetic alterations associated with the grade of astrocytic tumor by comparative genomic hybridization

A method of induction of multiple DNA-protein and protein-protein cross-links in chromatin of isolated nuclei has been developed. The cross-linking is brought about by mutual effect of fluorochrome ethidium bromide (EtBr) and irradiation with blue light. Electron microscopic analysis of nuclei isolated in solutions with various concentrations of divalent cations, stained with EtBr and irradiated with blue light, has demonstrated that the method leads to a selective stabilization of macromolecular complexes of chromatin against various treatments causing decompactization of native chromatin. Besides that, the stabilization of nucleoli and clusters of interchromatin granules takes place. On the other hand, the same treatment does not stabilize elementary chromatin fibers (about 30 nm thick), and the transition to the nucleosomal fiber occurs after extraction of histone H1 with 0.6 M NaCl. Electrophoretic analysis of proteins from control and irradiated nuclei shows the basic role of non-histone proteins in the stabilization. The data are discussed based on the assumption on the availability of some "intermediate" levels of chromatin compaction between 30 nm chromatin fiber and mitotic chromosome.     

Enzyme-linked immunosorbent assay for use with nuclear protein-DNA complex antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for identification and quantification of nuclear antigens (nonhistone protein-DNA complexes from chromatin). Until now, the complement fixation assay has been the only immunoassay routinely applied to nonhistone protein-DNA complexes. The ELISA is considerably more sensitive than the micro-complement fixation test for assaying the immunospecificity of nuclear protein-DNA complexes. Dilutions of rabbit antisera as great as 1:6400 could be used to detect nanogram quantities of antigen, chicken reticulocyte chromatin or dehistonized rat liver chromatin.     

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.     

Mode of reconstitution of chicken erythrocyte and reticulocyte chromatin

The mode of reconstitution of chicken erythrocyte and reticulocyte chromatin has been investigated. Chromatin was dissociated in 2 M NaCl, 5 M urea, and 0.01 M potassium phosphate (pH 7.2) and was dialyzed against various NaCl concentrations in 5 M urea and 0.01 M potassium phosphate (pH 7.2). Histone reassociation to DNA occurs with the binding of histone H5 at 0.5 M NaCl in 5 M urea, followed by histone H1 at 0.4 M NaCl in 5 M urea. All the classes of histones are reassociated with DNA at 0.2 M NaCl in 5 M urea and binding of all classes of histones is complete in 0.1 M NaCl and 5 M urea. Nonhistone proteins reassociate with DNA before and at the same time that histones reassociate with DNA. Binding of nonhistone proteins to DNA appears to be complete in 5 M urea and 0.01 M potassium phosphate (pH 7.2). There is also found in both erythrocyte and reticulocyte chromatin a nonhistone protein present in relatively high concentrations, which remains associated with DNA in 2 M NaCl and 5 M urea. This tightly bound protein appears as one major band when chromatographed on sodium dodecyl sulfate-polyacrylamide gels, with a molecular weight of 95 000. This protein is soluble in phenol and sodium dodecyl sulfate but is insoluble in 5 M urea or 4 M guanidine hydrochloride. A fraction of reticulocyte nonhistone proteins was found to bind to DNA-cellulose in 5 M urea. The majority of these proteins elute at 0.15 M NaCl in 5 M urea but a significant fraction elutes at NaCl concentrations at which the bulk of the histones do not bind to DNA. The proteins that bind to free DNA have low molecular weights and do not show species speciificity. Approximatley 50% of the reticulocyte nonhistone protein does not bind to a DNA-cellulose column in 5 M urea and may require histones for complete reassociation.     

Conformational studies of a synthetic peptide from the putative lipid-binding domain of bovine milk component PP3

In bovine milk, a glycosylated phosphoprotein, component PP3, is known for its remarkable emulsifying properties and its capability to inhibit lipolytic activities. The determination of its primary structure is not sufficient to explain these properties. Secondary structure predictions of component PP3 and of its homologous proteins were achieved using a combination of multiple predictive methods. Based on this study, the f 119-135 region of component PP3 was proposed to be likely to adopt an amphipathic helical conformation, which is a lipid-binding motif. The conformation of the synthetic peptide corresponding to the C-terminal f 119-135 part of bovine component PP3 was analyzed by circular dichroism experiments using various media. The circular dichroism data indicated that the peptide was able to form an amphipathic alpha-helix structure in trifluoroethanol as well as in the presence of sodium dodecyl sulfate or acidic and neutral lipids, but not in water. Moreover, the conformation of this peptide is solvent dependent because it was found to adopt a beta-sheet structure for low concentrations of sodium dodecyl sulfate or a low molar ratio of acidic lipid to peptide. Tensiometric measurements showed that the amphipathic C-terminal region of component PP3 is highly tensioactive and, thus, must be responsible for the particular behavior of the protein in emulsions.     

Temperature-induced conformational transition of intestinal fatty acid binding protein enhancing ligand binding: a functional, spectroscopic, and molecular modeling study

Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between 35 and 50 degreesC, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55) decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), this phenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acid occurs. Nevertheless, other spectroscopic evidence indicates that the beta-barrel structure, the major motif of this protein, is highly stable up to 70 degreesC. No changes associated with conformation were detected for both structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experiments in which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and its dimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereas that of bisANS increases slightly in the temperature range of 35-50 degreesC. Given the different size of these probes, model building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influences the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].     

Physicochemical properties of calcium phosphate coatings produced by pulsed laser deposition at different water vapour pressures

Conformational peculiarities of DNA complexes with histones of the H1 family have been studied by the method of circular dichroism (CD). The H1 histones were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius, starfish Aphelasterias japonica, and bivalve mollusc Chlamis islandicus and also from the rat thymus. It is shown that these sperm-specific histones do not compact DNA in low ionic strength solution. At physiologic conditions H1 from the sea urchin and starfish sperms compact DNA more intensively than other histones. The H1 from rat thymus has a minimum ability to compact DNA. This histone does not change the structure of DNA double helix. It was supposed that it could be associated with interactions of this histone with DNA in the major groove of its helix. At the same time sperm-specific H1 can interact with DNA not only in the major groove but also in the minor groove, and this induces changes in DNA structure. This DNA-protein interaction is specific for the sperm chromatin and may support the supercompact organization of the sperm chromatin.     

Transvaginal repair of vault prolapse: a review

The effect of histone acetylation was monitored on CHO chromatin structure, following the addition of 7 mM Na-butyrate to the cell culture medium. The properties of both control and hyperacetylated chromatins and nuclei were investigated by circular dichroism, ethidium bromide intercalation, differential scanning calorimetry, and affinity chromatography. Our results are compatible with modest but significant alterations in the various levels of chromatin organization, as a result of the charge neutralization of some lysine residues within the N-terminal region of the histonic octamer. Namely, large statistically significant differences do exist in the heat capacity thermograms of native nuclei, where unfolding into single nucleofilament of the highly packed native chromatin superfiber appears associated with acetylation; at the same time CD, EB, and affinity chromatography point to modest but consistent differences in the compactness of isolated nucleosomes and polynucleosomes.     

The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy

The kinetics of the guanidine hydrochloride-induced unfolding and refolding of bovine beta-lactoglobulin, a predominantly beta-sheet protein in the native state, have been studied by stopped-flow circular dichroism and absorption measurements at pH 3.2 and 4.5 degrees C. The refolding reaction was a complex process composed of different kinetic phases, while the unfolding was a single-phase reaction. Most notably, a burst-phase intermediate of refolding, which was formed during the dead time of stopped-flow measurements (approximately 18 ms), showed more intense ellipticity signals in the peptide region below 240 nm than the native state, yielding overshoot behavior in the refolding curves. We have investigated the spectral properties and structural stability of the burst-phase intermediate and also the structural properties in the unfolded state in 4.0 M guanidine hydrochloride of the protein and its disulfide-cleaved derivative. The main conclusions are: (1) the more intense ellipticity of the intermediate in the peptide region arises from formation of non-native alpha-helical structure in the intermediate, apparently suggesting that the folding of beta-lactoglobulin is not represented by a simple sequential mechanism. (2) The burst-phase intermediate has, however, a number of properties in common with the folding intermediates or with the molten globule states of other globular proteins whose folding reactions are known to be represented by the sequential model. These properties include: the presence of the secondary structure without the specific tertiary structure; formation of a hydrophobic core; broad unfolding transition of the intermediate; and rapidity of formation of the intermediate. The burst-phase intermediate of beta-lactoglobulin is thus classified as the same species as the molten globule state. (3) The circular dichroism spectra of beta-lactoglobulin and its disulfide-cleaved derivative in 4.0 M guanidine hydrochloride suggests the presence of the residual beta-structure in the unfolded state and the stabilization of the beta-structure by disulfide bonds. Thus; if this residual beta-structure is part of the native beta-structure and forms a folding initiation site, the folding reaction of beta-lactoglobulin may not necessarily be inconsistent with the sequential model. The non-native alpha-helices in the burst-phase intermediate may be formed in an immature part of the protein molecule because of the local alpha-helical propensity in this part.     

Effects of potassium chloride and temperature on the kinetics of recombination of heavy and light chains of a human IgGl myeloma protein

The effects of KCl and temperature on the kinetics of the recombination reaction of alkylated H and L chains of a human myeloma protein were studied by means of circular dichroism (CD). The recombination rate was considerably reduced by the presence of KCl. The results of the difference spectra showed that tryptophyl residue(s) in L chains are perturbed by KCl. No changes in the CD and absorption spectra with KCl concentration were observed for H chains and recombined H2L2. The change in the rate constant with KCl concentration paralleled the changes in the CD at 235 nm and the difference in molar extinction coefficient at 293 nm. These facts suggest that the reduction of the recombination rate in the presence of KCl is due to a change in the conformation of L chains as a result of specific interaction with chloride ions or by a shift to the dimer form in the monomer-dimer equilibrium of L chains. The rate constant showed a temperature dependence with an activation energy of 17.4 kcal/mole.     

Scanning electron microscopy of the tunica albuginea of the corpora cavernosa in normal and impotent subjects

Structural effects of glycosylation of the C-terminal pentapeptide fragment of Peptide T (Thr-Thr-Asn-Tyr-Thr) were studied. Because of the inherent flexibility of these molecules, molecular simulations are used to interpret the circular dichroism and nuclear magnetic resonance data acquired for these molecules. N-acetyl-glucosamine attached at the Asn residue changes the ensemble average backbone conformation of the peptide and limits the conformational space available to the pentapeptide fragment. Glycosylation changes the type I and III beta-turn propensity of the pentapeptide to a type II turn. Since glycosylation also increases peptide solubility and inhibits peptide degradation in human serum, glycopeptide design may be an efficient approach to stabilize or conformationally modify peptide drug candidates and to create additional diversity in peptide libraries.