Hana Votavová

Changes in Conformation, Stability and Condensation of DNA by Univalent and Divalent Cations in Methanol-Water Mixtures

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.

High-performance liquid chromatographic enantiomeric resolution in the ten-vertex carborane series: Comparison of acetyl- and native β-cyclodextrin bonded chiral stationary phases

The HPLC resolution on native and acetyl β-cyclodextrin columns is reported for thirteen enantiomeric pairs of racemates, members of two series of positional isomers in the ten-vertex carborane family, the exo-9-L-arachno-5,6-C2B8H12 (L=NH3, primary or secondary amino group) or exo-6-L-arachno-5,10-C2B8H12 (L=secondary or tertiary amino group). The study of influence of the structural factors on retention, selectivity and resolution of individual compounds is described on both chiral stationary phases (CSPs). The deep differences in enantio-selectivities has been observed for native and acetyl β-cyclodextrin CSPs. Circular dichroism (CD) spectra of exo-9-t-BuNH2-arachno-5,6-C2B8H12 and exo-6-Et3N-arachno-5,10-C2B8H12, members of each series are presented. The CD results indicate the different enantiodiscrimination mechanism on the two CSPs under study.

Selective Binding of Synthetic Polypeptides to DNA of Varying Composition and Sequence: Effect of Minor Groove Binding Drugs

Repetitive basic polypeptides containing lysine or arginine as every third amino acid were shown to cause DNA condensation at physiological salt concentration connected with selective DNA binding with respect to DNA composition and sequence. This selectivity is very similar to that existing in the case of histone H1 and other basic proteins and does not depend on polypeptide chain conformation. The effect of the minor groove binding drugs netropsin and distamycin was tested to elucidate the origin of the binding selectivity. The results suggest that the binding preferences are due to the variations in the conformation in various types of B-DNA that depend on DNA composition and sequence. The most important factor affecting the selectivity is probably the value of the negative electrostatic potential in the minor groove.

Interaction of a bZip Oligopeptide Model With Oligodeoxyribonucleotides Modelling DNA Binding Sites. The Effect of Flanking Sequences

A leucine zipper (bZip) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZip proteins (Jun) with related DNA binding specificity. Self-complementary DNA hexadecanucleotides containing ATF/CRE, AP-1 and C/EPB target sites were used to study peptide-DNA complex formation. Conformation changes in both components that occur on complex formation were studied by circular dichroism (CD) spectroscopy. The results show that the amount of alpha-helix formed in the peptide strongly depends not only on the target site present, but also on the type of the sequence flanking the ATF/CRE target site. Highest amount of the alpha-helix induced in the peptide was observed when homopurine homopyrimidine flanking sequences were present, whereas the presence of alternating sequences, especially of the CA/TG type, showed considerably lower effects. The change in DNA conformation on complex formation was generally small, but also depended on the type of the flanking sequence. It appears that the sequences flanking the target site can considerably modify the ability of the target sequence to bind specifically the bZip peptide, probably by slightly varying the overall DNA conformation.

Synthesis and structure of 6-amino-2,3,6-trideoxy-d-erythro-hexono-1,6-lactam and 6-amino-3,6-dideoxy-d-xylo-hexono-1,6-lactam

Solid-state conformations of 6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3a) and 6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7a) were determined using X-ray diffraction. Conformations of the compounds 3a, 7a, and their per-O-acetyl derivatives 4,5-di-O-acetyl-6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3b) and 2,4,5-tri-O-acetyl-6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7b) in solutions were deduced from the analysis of NMR spectra using a modified Karplus equation and compared with the results of circular dichroism measurement of lactams 3a and 7a. Conformation 4C(1,N) was revealed for solid lactams 3a and 7a and for lactams 7a and 7b in solution, while lactams 3a and 3b in solution exist in the approximately 1:1 equilibrium of the conformers 4C(1,N) and (1,N)C4.

Synthesis and chiroptical properties of some abbreviated NAD+ analogues

Abstract Four novel “abbreviated” NAD + analogues were prepared by the Zincke reaction. The acyclic chain joining the bases of the analogues bears two stereogenic centers with hydroxyl groups in the both erythro and threo mutual relations. The CD spectra of all stereoisomers were measured and discussed.

CD studies of interaction of a bZIP oligopeptide model with DNA

Abstract A leucine zipper (bZIP) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZIP proteins (Jun) with related DNA binding specificity. Selfcomplementary DNA hexadecanucleotides containing modified ATF/CRE target sites were used to study peptide–DNA complex formation. Four oligonucleotides contained substitutions of two GC or AT pairs by IC pairs in the ATF/CRE target sequence. In two other oligonucleotides there was a substitution of A by I in two AT pairs (mismatch IT pairs were presumably formed in the duplex) and one oligonucleotide contained I instead of C in two base pairs (IG mismatch in the duplex). Conformation changes of BP1 that occur on complex formation were studied by circular dichroism spectroscopy. The binding of peptide BP1 to oligonucleotides is accompanied by an increase of the α-helix content, which depends strongly on the oligonucleotide sequence. The substitution of two GC pairs within the specific binding site has either none or only a small effect. However, the substitution of two AT pairs within the binding site by IC strongly decreases the specificity of binding to a level observed with an oligonucleotide containing the C/EBP binding site, differing from the ATF/CRE site at four positions (Votavova et al., J. Biomol. Struct. Dyn. 3 (1997) 587). Similar results were obtained also with an oligonucleotide containing I instead of C in two base pairs (IG mismatch in the duplex). Two oligonucleotides with two substitutions of A by I but with unchanged T in the AT pairs (IT mismatch) showed smaller decrease in the α-helix formation on peptide binding than oligonucleotides, in which the whole AT pair was replaced by IC. The effect of such a substitution depends on the position of the original AT pairs in the target sequence, but the presence of T appears to be essential for specific peptide binding.

NATURE OF PROTEIN – DNA INTERACTIONS REVEALED BY MODEL POLYPEPTIDE COMPLEXES

Publisher Summary Generally, two types of DNA - binding proteins can be recognized: (1) specific binding proteins, for example, repressors and other regulatory proteins and (2) nonspecific binding proteins, for example, histones or protamines. The primary and higher structures of the entire protein molecule may affect the binding to DNA. It is assumed that the binding is largely determined by the structure of the binding polypeptide segment. Basic polypeptides are designed and synthesized with controlled amino acid composition and sequence to study the effect of polypeptide primary structure and conformation on the binding to DNA. The affinity of polypeptides to DNA and the mechanism of binding are substantially affected by two factors in the polypeptide primary structure: the nature, content, and distribution of basic residues and, the presence of strongly hydrophobic residues.