Nuclear magnetic resonance studies on the solution conformation of histone IV fragments obtained by cyanogen bromide cleavage.

Two histone IV fragments obtained by cleavage at Met-84 by cyanogen bromide have been examined by proton magnetic resonance (PMR) spectroscopy as a function of temperature, peptide concentration, ionic strength, and pD. Sedimentation and gel electrophoresis studies on these peptides have also been carried out. The 220-MHz PMR spectrum of the N-peptide in both the high- and low-field regions was shown to be almost identical with that calculated for an extended coil N-peptide monomer. The calculated random coil and experimental C-peptide spectra, on the other hand, differ in many respects. Evidence was obtained for the presence of rigid secondary structure in the C-peptide. In addition, the Val, Leu, Ile CH3 resonance displays a prominent high-field satellite band which shifts downfield with increasing temperature. Sedimentation studies on the N-peptide reveal the formation of extremely large, remarkably homogeneous aggregates at ionic strengths larger than or equal to 0.01. The C-peptide, on the other hand, does not appear to form aggregates of sufficient size to be detectable in velocity sedimentation studies of a few hours duration. The relative area changes which have previously been noted in the PMR spectrum of histone IV with increasing ionic strength were also observed for the N-peptide but not the C-peptide. Interpretation of these relative area changes has been made in terms of the amino acid sequence of histone IV, and an effort was made to identify that segment of the polypeptide which undergoes secondary structural change with increasing ionic strength.     

Self-association of puromycin as studied by proton magnetic resonance spectroscopy

The self-association of puromycin has been studied using proton magnetic resonance spectroscopy. The concentration, temperature and pH dependence studies of the proton chemical shifts of the adenine protons indicate that puromycin in aqueous solution at pD 7.4 self associates predominantly through adenine-adenine interaction. At this pD, the amino group of the aminoacyl segment of puromycin has been demonstrated to exist in a equilibrium blend of protonated and non-protonated forms. At pD 2.6, PM is found to exist predominantly in the monomeric from in which the methyl groups of the 6N-dimethyladenine are found to be non-equivalent due to hindered rotation about the C6-N6 bond.     

Self-Association of Puromycin as studied by Proton Magnetic Resonance Spectroscopy

Abstract

The self-association of puromycin has been studied using proton magnetic resonance spectroscopy. The concentration, temperature and pH dependence studies of the proton chemical shifts of the adenine protons indicate that puromycin in aqueous solution at pD 7.4 self associates predominantly through adenine-adenine interaction. At this pD, the amino group of the aminoacyl segment of puromycin has been demonstrated to exist in a equilibrium blend of protonated and non-protonated forms. At pD 2.6, PM is found to exist predominantly in the monomeric from in which the methyl groups of the 6N-dimethyladenine are found to be non-equivalent due to hindered rotation about the C6-N6 bond.     

Proton magnetic resonance study of complex formation between N6-methyladenosine and polyuridylic acid

The interaction between N₆-methyladenosine and polyuridylic acid in D₂O solution at neutral pD has been studied as a function of temperature and N₆-methyladenosine concentration by proton magnetic resonance spectroscopy. A rigid double-stranded 1:1 complex is formed below ～10°C, involving hydrogen-bonded N₆-methyladenine:uracil base-pairing and stacking of the adenine bases. This complex is less stable than the 1:2 complex formed between adenosine and polyU, and involves a more rapid exchange of the monomer between free and polymer-bound environments.     

The secondary structure of histone IV and its stability.

The secondary structure within histone IV and its fragments obtained by cyanogen bromide (CNBr) and cleavage at Met 84 has been examined by circular dichroism and spectophotometric pH titration measurements. These studies have confirmed the existence of stable secondary structure within the C-terminal fragment of histone IV (C-peptide which can be perturbed only by 6M urea at pH greater than 8 or 8 M guanidine-HCL. In contrast, the N-terminal fragment (N-peptide) appears to lack significant secondary structure at low ionic strengths but acquires approximately 15% betasheet conformation and 5% alpha-helix upon aggregation at ionic strengths larger than or equal to 0.4. The rates of nitration of the N- and C-peptides by tetranitromethane (TNM) have also been measured as a function of ionic strengths. Under comparable conditions, the rate constant for nitration of the N-peptide was found to be about six times greater than that for the C-peptide, further evidence in support of the presence of stable secondary structure within the C-terminal region of histone IV. After binding these histone IV fragments to DNA, however, the nitration reaction rate constants for the N- and C-peptide in the bound form are found to be 2% and 27% of the corresponding free peptides. Reconstituted nucleohistone IV is about 10% as reactive to TNM as histone IV at comparable ionic strength.     

Effects of molecular association on structure and dynamics of a collagenous peptide

Peptide GVKGDKGNPGWPGAPY from the triple-helix domain of type IV collagen aggregates in solution at a critical aggregation concentration of 18 mM. This molecular self association process is investigated by ¹H- and ¹³C-nmr spectroscopy. As a function of increasing peptide concentration, selective ¹H resonances are cooperatively chemically shifted by up to 0.04 ppm to apparently saturable values at high concentration. Pulsed field gradient nmr was used to derive translation diffusion constants that, as the peptide concentration is increased, also cooperatively and monotonically decrease to an apparent limiting value. An average number of 6 monomer units per aggregate have been estimated from diffusion constant and ¹³C relaxation data. Comparative ¹H nuclear Overhauser effect spectroscopy (NOESY) spectra accumulated at high and low peptide concentrations suggest that average internuclear distances are decreased as a result of peptide association. ¹³C-nmr multiplet spin-lattice relaxation and ¹³C- {¹H} NOE effects on ¹³C-enriched glycine methylene positions in the peptide demonstrate that overall molecular tumbling and backbone internal motions are attenuated in the aggregate state. Lowering the solution pD from pD 6 to pD 2 disrupts the aggregate state, suggesting a role for electrostatic interactions in the association process. Based on thermodynamic considerations, hydrophobic interactions also probably act to stabilize the aggregate state. These data are discussed in terms of an nmr/NOE constrained computer-modeled structure of the peptide. © 1993 John Wiley & Sons, Inc.     

A pH-dependent interaction between histones H2A and H2B involving secondary and tertiary folding.

It has been shown by high-resolution proton magnetic resonance (PMR) spectroscopy and circular dichroism (CD) that an H2A/H2B histone complex exists after salt extraction of these histones from chromatin and that this complex can be fully renatured from both urea-denatured acid-extracted and from urea-denatured salt-extracted histones. The histone complex is shown to involve specific secondary and tertiary structure. Formation of this complex is observed to be critically dependent on pH, occurring at and above pH 5. It cannot be induced below pH 5 by increase in ionic strength. From CD spectra the H2A/H2B complex is shown to contain about 37% alpha helix but no beta structure, the latter being confirmed by infrared spectroscopy in the 6-mum region. The PMR spectra show that the structured region includes most of the aromatic residues of both histones, at least two histidine residues of H2B and probably histidines 31 and 82 of histone H2A. The secondary structure of histones H2A and H2B is predicted using the Chou and Fasman procedure and comparisons are made between the predictions for histones of different species. These results in conjunction with the experimental evidence lead to the conclusion that at least residues 31-95 of H2A and residues 37-114 of H2B, i.e. the more apolar regions of the molecules, are involved in the tertiary structure of the H2A/H2B complex.     

Interaction of thiamine diphosphate with magnesium ion

The action of magnesium ion on the exchange rate of the proton in C2 of thiamine and thiamine diphosphate is studied at different values of pD. Above pD 5 the ion Mg2+ increases this exchange rate. The phenomenon is markedly enhanced for TDP rather than thiamine and increases with pD. Below pD 5 magnesium decreases the exchange rate. This decrease is greater for TDP than for thiamine. The maximum effect is reached at a magnesium concentration of 0.5/1 for thiamine and of 1/1 for TDP. T1 measurements are made for different pH values with and without magnesium ion. Results seem to prove that an increase in pD values from 3.9 to 5.9 leads to an accentuation of the molecules "folded" form. Nevertheless for a given pD value the TDP-Mg complex seems to have a more "folded" form than TDP.     

Comparison of membrane organization in mitochondria from yeast and rat liver by nuclear magnetic resonance spectroscopy

Summary Proton magnetic resonance (PMR) and carbon-13 magnetic resonance (CMR) spectra of intact, unsonicated yeast and rat liver motochondria show differences which may be correlated with the composition of the membranes. High resolution PMR and CMR signals in intact yeast mitochondria have been assigned to regions of fluid lipid-lipid interaction on the basis of spectra of extracted lipid and protein, and the temperature dependence of NMR signals from the intact membrane. PMR spectra suggest that about 20% of total yeast phospholipid is in regions where both intramolecular fatty acid chain mobility and lateral diffusion of entire phospholipid molecules are possible. No such regions appear to exist in rat liver mitochondria. For both yeast and rat liver mitochondria, comparison of PMR and CMR spectra suggests that about 50% of phospholipid appears to be in regions where intramolecular fatty acid chain motion is considerable, but lateral diffusion is restricted. The remaining phospholipid appears to have little inter- or intramolecular mobility. Since NMR observation of lipid extracts from membranes indicates that phospholipid-sterol interactions do not account for the spectra of intact mitochondria, these effects are interpreted in terms of extensive lipid-protein interactions.     

Necleoside conformations. 19. Temperature and pH effects on the conformation of guanosine phosphates.

Proton magnetic resonance spectra at 250 MHz were measured as a function of temperature and pH of the three guanosine phosphates. From these data and previously published work the conformational parameters of these compounds were determinated. The phosphate group of Guo-5'-P changes its conformation around the C-O bond and its rotation is relatively slow at 20 degrees. At neutral pD the S conformation is favoured and the N form at acid pD. This conformational change is paralleled by a change in exocyclic rotamer distribution and takes place at the pK of the protonation of the base on N-7. Although correlation appears to exist between the various conformations, notable exceptions exist.     

Physical properties of inner histone-DNA complexes.

Chicken-erythrocyte inner histone tetramer has been complexed with several natural and synthetic DNA duplexes by salt-gradient dialysis at various protein/DNA ratios. The resulting complexes, in low-ionic-strength buffer, have been examined by electron microscopy, circular dichroism, and thermal denaturation. Electron microscopy reveals nucleosomes (nu bodies) randomly arranged along DNA fibers, including poly(dA-dT)-poly(dA-dT), poly(dI-dC)-poly(dI-dC), but not poly(dA)-poly(dT). Circular dichroism studies showed prominent histone alpha-helix and "suppression" of nucleic acid ellipticity (lambda less than 240 nm). Thermal denaturation experiments revealed Tm behavior comparable to that of H1- (or H5-) depleted chromatin. Tm III and Tm IV increased linearly with G + C%(natural DNAs), but were virtually independent of the histone/DNA ratio; therefore, the melting of nucleosomes along a DNA chain is insensitive to adjacent "spacer" DNA lengths. This suggests that Tm III and Tm IV arise from the melting of different domains of DNA associated with the core nu body.     

Effects of pD on ring-stacking interactions between dinucleoside phosphates and tryptamine

Complex formation between tryptamine and mononucleotides and dinucleoside phosphates containing adenine and/or cytosine has been studied at five pD's ranging from 1.1 to 7.4 by proton magnetic resonance spectroscopy. Chemical shifts of base ring protons and the ribose anomeric proton in the nucleotides and indole ring protons in tryptamine were monitored and their changes with pD and intermolecular interactions interpreted qualitatively. Stacked complexes were found to exist at all pD's in the range studied. Complex geometries differ depending on pD. An electrostatic interaction between the tryptamine amino group and the nucleotide phosphate group contributes to complex formation above pD 4 but is not strong enough to shift the dinucleoside phosphate equilibrium towards the unstacked conformer.     

Grafting of oligosaccharides onto synthetic polymer colloids

A new method to form colloidally stable oligosaccharide-grafted synthetic polymer particles has been developed. The oligosaccharides, of weight-average degree of polymerization approximately 38, were obtained by enzymatic debranching of amylopectin. Through the use of a cerium(IV)-based redox initiation process, oligosaccharide chains are grafted onto a synthetic polymer colloid comprising electrostatically stabilized poly(methyl methacrylate) or polystyrene latex particles swollen with methyl methacrylate monomer. Ce(IV) creates a radical species on these oligosaccharides, which then propagates, initially with aqueous-phase monomer, then with the methyl methacrylate monomer inside the particles. Ultracentrifugation, NMR, and total starch analyses together prove that the grafting process has occurred, with at least 7.7 wt % starch grafted and a grafting efficiency of 33%. The surfactant used in latex preparation was removed by dialysis, resulting in particles colloidally stabilized with only linear starch as a steric stabilizer. The debranched starch that comprises these oligosaccharides is found to be a remarkably effective colloidal stabilizer, albeit at low electrolyte concentration, stabilizing particles with very sparse surface coverage.     

Comparison of the heme structures of horseradish peroxidase compounds X and II by resonance Raman spectroscopy

Horseradish peroxidase will catalyze the chlorination of certain substrates by sodium chlorite through an intermediate known as compound X. A chlorite-derived chlorine atom is known to be retained by compound X and has been proposed to be located at the heme active site. Although several heme structures have been proposed for compound X, including an Fe(IV)-OCl group, preliminary data previously reported by our laboratory suggested that compound X contained a heme Fe(IV) = O group, based on the similarity of a compound X resonance Raman band at 788 cm-1 to resonance Raman Fe(IV) = O stretching vibrations recently identified for horseradish peroxidase compound II and ferryl myoglobin. Isotopic studies now confirm that the 788 cm-1 resonance Raman band of compound X is, in fact, due to a heme Fe(IV) = O group, with the oxygen atom derived from chlorite. The Fe(IV) = O frequency of compound X, of horseradish peroxidase isoenzymes B and C, undergoes a pH-induced frequency shift, with behavior which appears to be the same as that previously reported for compound II, formed from the same isoenzymes. These observations strongly suggest that compounds II and X have very similar, if not identical, heme structures. The chlorine atom thus appears not to be heme-bound and may rather be located on an amino acid residue. The studies on compound X reported here were done in a pH region above pH 8, where compound X is moderately stable. The present results do not necessarily apply to compound X below pH 8.     

Studies on the reactive properties of histone amino groups: reactivities of free histones and histones in chromatin as a function of ionic strength.

The reactivity of the amino groups of the five histones towards acetic anhydride has been measured and with the exception of histone IIb2 the reactivities are very similar to those of exposed lysines with an average pK of 9.5. In addition the reactivities of these groups from 0.20 to 1.0 M NaCl and the reactivity of a peptide containing lysines 5, 8, 12 and 16 of histone IV have been measured in chromatin. It is concluded that at the lower ionic strengths the large proportion of the amino groups are buried for both the histones and the region of histone IV studied. Data obtained from the measurement of the reactivity of standard proline compounds and from a pH and ionic strength study indicate that the N-terminal proline of histone IIb2 is exposed.     

An abnormal pK(a) value of internal histidine of the insulin molecule revealed by neutron crystallographic analysis

Insulin is stored in pancreatic β-cell as hexameric form with Zn²⁺ ions, while the hormonally active form is monomer. The hexamer requires the coordination of Zn²⁺ ions to the HisB10. In order to reveal the mechanism of the hexamerization of insulin, we investigated the Zn²⁺ free insulin at pD6.6 and pD9 by neutron crystallographic analyses. HisB10 is doubly protonated not only at pD6.6 but also at pD9, indicating an abnormal pK_a of this histidine. It is suggested that HisB10 acts on a strong cation capture and contributes to the high stability of the hexameric form in pancreas.     

Analysis of pD-dependent complexation of N-benzyloxycarbonylaminophosphonic acids by alpha-cyclodextrin. Enantiodifferentiation of phosphonic acid pKa values

The influence of aqueous solution pD on stereoselective complexation of N-benzyloxycarbonylaminophosphonic acids with alpha-cyclodextrin was investigated by means of nuclear magnetic resonance spectroscopy. The highest enantiodiscrimination was achieved at pD close to the pKa of less acidic hydroxyl group of the phosphonic moiety of analytes (6.5-7.5). This effect results from the stereoselective differentiation of pKa (up to 0.28 pD unit) upon complexation with applied chemical shift reagent. Moreover, analysis of 2D-ROESY spectra proved that the host-guest inclusion mode is strongly influenced by pD.     

A spectroscopic and thermodynamic study of porphyrin/DNA supramolecular assemblies.

Assemblies of trans-bis(N-methylpyridinium-4-yl)diphenylporphine ions on the surface of calf thymus DNA have been studied using several spectroscopic techniques: absorbance, circular dichroism, and resonance light scattering. The aggregation equilibrium can be treated as a two-state system-monomer and assembly-each bound to the nucleic acid template. The aggregate absorption spectrum in the Soret region is resolved into two bands of Lorentzian line shape, while the DNA-bound monomer spectrum in this region is composed of two Gaussian bands. The Beer-Lambert law is obeyed by both porphyrin forms. The assembly is also characterized by an extremely large, bisignate induced circular dichroism (CD) profile and by enhanced resonance light scattering (RLS). Both the CD and RLS intensities depend linearly on aggregate concentration. The RLS result is consistent with a model for the aggregates as being either of a characteristic size or of a fixed distribution of sizes, independent of total porphyrin concentration or ionic strength. Above threshold values of concentration and ionic strength, the mass action expression for the equilibrium has a particularly simple form: K' = cac-1; where cac is defined as the "critical assembly concentration."offe dependence of the cac upon temperature and ionic strength (NaCl) has been investigated at a fixed DNA concentration. The value of the cac scales as the inverse square of the sodium chloride concentration and, from temperature dependence studies, the aggregation process is shown to be exothermic.