Conformational study on poly [gamma-(alpha-phenethyl)-L-glutamate] using vibrational circular dichroism spectroscopy.

Vibrational circular dichroism (VCD) and IR absorption spectra are obtained in a chloroform solution for poly[gamma-((R)-alpha-phenethyl)-L-glutamate] (PRPLG) and poly[gamma-((S)-alpha-phenethyl)-L-glutamate] (PSPLG), whose only structural difference is an opposite chiral center in the side chain. Their characteristic amide A, I, and II bands show VCD patterns quite similar to those of poly[gamma-benzyl-L-glutamate] (PBLG), indicating that the secondary structure of these polypeptides is a right-handed alpha-helix. The VCD spectra in the CH stretching region exhibit different patterns for PRPLG and PSPLG, reflecting the chirality difference in the side chains. This difference is interpreted on the basis of the additivity of optical activity contributions from the main chain conformation and the chirality difference in the side chains. The results indicate that a VCD difference spectrum of the CH stretching region is a useful diagnostic tool for elucidating local chirality differences.

Determination of the conformation of d(GGAAATTTCC)2 in solution by use of 1H NMR and restrained molecular dynamics.

The conformation of the putative bent DNA d(GGAAATTTCC)2 in solution was studied by use of 1H NMR and restrained molecular dynamics. Most of the resonances were assigned sequentially. A total of 182 interproton distance restraints were determined from two-dimensional nuclear Overhauser effect spectra with short mixing times. Torsion angle restraints for each sugar moiety were determined by qualitative analysis of a two-dimensional correlated spectrum. Restrained molecular dynamics was carried out with the interproton distances and torsion angles incorporated into the total energy function of the system in the form of effective potential terms. As initial conformations for restrained molecular dynamics, classical A-DNA and B-DNA were adopted. The root mean square deviation (rmsd) between these two conformations is 5.5 A. The conformations obtained by use of restrained molecular dynamics are very similar to each other, the rmsd being 0.8 A. On the other hand, the conformations obtained by use of molecular dynamics without experimental restraints or restrained energy minimization depended heavily on the initial conformations, and convergence to a similar conformation was not attained. The conformation obtained by use of restrained molecular dynamics exhibits a few remarkable features. The second G residue takes on the BII conformation [Fratini, A. V., Kopka, M. L., Drew, H. R., & Dickerson, R. E. (1982) J. Biol. Chem. 257, 14686-14707] rather than the standard BI conformation. There is discontinuity of the sugar puckering between the eighth T and ninth C. The minor groove of the oligo(dA) tract is rather compressed. As a result, d(GGAAATTTCC)2 is bent.

A new model for the bending of DNAs containing the oligo(dA) tracts based on NMR observations.

The conformations of double-stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) and d(GCATTTTGAAACG)d(CGTTTCAAAATGC) have been studied by NMR spectroscopy. Analyses of cross peaks in NOESY spectra between the H2 of an adenine and the H1' of a deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' in each duplex. In view of this gradual compression of the minor groove along the oligo(dA) tract, it can be understood clearly why d(GGAAATTTCC)n x 2 and d(GAAAATTTTC)n x 2 are bent, and d(GGTTTAAACC)n x 2 and d(GTTTTAAAAC)n x 2 are not bent. The relative extents of bending of a series of d(AjN10-j)nd(N10-jTj)n sequences can also be understood systematically. Additionally, it was found that the TA step disturbed the compression of the minor groove of the oligo(dA) tract to some extent.

A new model for DNA bending on the basis of gradual compression of the minor groove from 5' to 3' along the oligo(dA) tract.

The conformations of double stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG).d(CGCTTTTTTGCG) and d(GCATTTTGAAACG).d(CGTTTCAAAATGC) have been studied by means of NMR spectroscopy. Analyses of cross peaks in NOESY spectra between H2 of an adenine and H1' of the deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' along the tract in four oligonucleotides. A new model is proposed as to DNA bending based on the evidence of gradual compression of the minor groove. This model can explain why d(GGAAATTTCC) n x 2 and d(GAAAATTTTC) n x 2 are bent, and d(GGTTTAAACC) n x 2 and d(GTTTTAAAAC) n x 2 are not bent. The bending of d(AjN10-j) n x 2 sequences can also be explained.

One- and two-dimensional NMR studies on the conformation of DNA containing the oligo(dA)oligo(dT) tract.

The resonances of the imino protons and all of the non-exchangeable protons (except for H5'/H5'') of d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) have been assigned by means of one- and two-dimensional NMR spectroscopies. Qualitative analyses showed that the overall structure is of the B-form, but local conformational deviations exist. The NOEs between the imino protons of thymines and H2 of adenines suggest that the A-T base pairs are propeller-twisted to almost the same degree as in crystals. A remarkable chemical shift of H1' was observed for the residue located just before the oligo(dA)oligo(dT) tract, suggesting the presence of conformational discontinuity at the junctions between the oligo(dA)oligo(dT) tract and the other portions. Analyses of cross peaks in NOESY spectra between H2 of adenines and H1' of the 3'-neighbouring residues on the complementary strand revealed that the minor groove of the oligo(dA)oligo(dT) tract is narrow and compressed gradually, from 5' to 3', along the tract.

Association of polyene antibiotics with sterols.

Molecular interaction between amphotericin B and sterols in non-aqueous solutions was examined quantitatively by spectroscopic methods in order to support the view point that selectivity of amphotericin B is more pronounced in the presence of ergosterol than of cholesterol. The most likely association complexes between ergosterol and amphotericin B are 4:1, 6:1 stoichiometric complex when the concentrations of amphotericin B are 3.93 x 10(-4) M, 1.94 x 10(-4) M respectively. The presence of 3 beta-OH group is necessary but not enough for the association with amphotericin B. It appears that the extent of spectral change of amphotericin B induced by complexing sterol is greater for ergosterol than cholesterol.

Determination of protonation sites in thermospermine and in some other polyamines by 15N and 13C nuclear magnetic resonance spectroscopy.

The pK values of thermospermine, a novel asymmetric tetraamine (4,8-diazadodecane-1,12-diamine), have been determined for the first time by the conventional titration method with subsequent data analyses. It was found to be the most basic polyamine among naturally occurring tetraamines. The 15N NMR titration method was applied to determine its protonation site. Natural-abundance 15N magnetic resonance spectra of the polyamine have been recorded as a function of pH from its free to the protonated form. Its apparent pK values, as well as the intrinsic chemical shifts for each stage of protonation, have been calculated from the NMR titration curve by using a nonlinear least-squares method. The four protonation sites of thermospermine have been successfully determined to be N12, N1, N-8, and N-4 in that order from the free to the completely protonated form. In a similar manner the protonation sites of spermidine and two symmetric tetraamines, thermine and spermine, have been established. 13C NMR experiments for the four polyamines were almost consistent with those of 15N NMR. Owing to the much higher concentration of the polyamines furnished for NMR titration experiments, the pK values obtained by NMR were larger than those derived by the conventional titration methods.

Detection of separated amino proton resonance signals of adenine derivatives of low temperature and its application to estimation of population of the adenine-uracil dimers in solution.

Splitting of the amino proton signals of 9-ethyladenine derivatives was found in proton nuclear magnetic resonance spectra at low temperature (ca. -30 degrees C). One of the separated signals corresponds to the syn amino proton relative to the N(1) nitrogen in the adenine ring and the other to the anti one. The phenomenon is ascribable to slowing down of the hindered rotation around the N(6)-C(6) bond, which has partial double bond character. On the addition of 1-cyclohexyluracil derivatives, one of the separated signals shifts downfield. From the analysis of the concentration dependence of the signals we could estimate the population of two kinds of adenine-uracil (AU) dimers that employ the syn and anti protons, respectively. i.e. the Watson-Crick-type and the Hoogsteen-type dimers. Independent of the substitution on the uracil ring, the Hoogsteen type is predominant at 70% and the Watson-Crick type at 30% (at -56 degrees C). On the other hand, with mixtures of general kinds of 9-ethyladenine derivatives with 1-cyclohexyluracil. the substituents on the adenine ring cause the population to deviate to extreme values; i.e., either the Watson-Crick-type or the Hoogsteen-type dimer predominates. 2-Chloro-9-ethyladenine and N2-(dimethylamino)-9-ethyladenine take almost completely the Hoogsteen-type dimers, while 8-bromo-9-ethyladenine, N2-(methylamino)-9-ethyladenine, and 2-amino-9-ethylpurine predominant in the Watson-Crick-type dimers.

Detection of proton-acceptor sites of hydrogen bonding in adenine X uracil base pairs by the use of 15N magnetic resonance.

2',3',5'-Tri-O-acetyl[1,3,7,9,amino-15N]adenosine(ac3Ado) and its 8-2H and 8-bromo derivatives (ac3[8-2H]Ado and ac3br8Ado) were synthesized from 95% 15N-enriched adenosine which was obtained by microbial fermentation. The chemical shifts and nuclear Overhauser effects of 15N NMR of the adenosine derivatives were measured by changing the concentration of the mixed 1-cyclohexyluracil (cHxUra) in chloroform. The limiting shift of each 15N resonance was calculated by using the association constants obtained from proton magnetic resonances of the ac3Ado/cHxUra and ac3br8Ado/cHxUra systems. From the quantitative analysis of the 15N chemical shifts of the N-1 and N-7 atoms it could be concluded that ac3Ado X cHxUra dimers prefer the Hoogsteen-type pair while the Watson-Crick-type pairs are predominant in the ac3br8Ado X cHxUra dimers. The N-3 atom, which does not locate at the interaction site, showed a fairly large induced shift. Thus the induced shift of 15N resonance by association does not always mean the involvement of the corresponding nucleus to the interaction site. The effect of intermolecular hydrogen bonding on the nuclear Overhauser enhancement of the 15N-7 resonance was observed in the ac3[8-2H]Ado/cHxUra mixture. The presence of the Hoogsteen-type hydrogen bonding was proposed by the use of 15N NMR spectra. The present information will be useful for the elucidation of non-Watson-Crick-type base-pair interaction in transfer TNA and oligonucleotides.

An 15N NMR study of adenine-uracil base pair in a non-aqueous solvent.

[1,3,7,9,10-15N]-2',3',5'-Tri-O-acetyl adenosine (A) and its 8-D and 8-Br derivatives (AD and ABr) were prepared from 95% 15N enriched adenosine obtained from microbial fermentation. The chemical shifts and nuclear Overhauser effects of 15N resonances were measured as a function of the concentration of the mixed 1-cyclohexyluracil. The limiting shift of each 15N resonance was calculated and the structure of the A-U dimer was estimated. From the shifts of 15N-1 and 15N-7 signals it is determined that ABr-U dimers prefer the Watson-Crick type hydrogen bond while the Hoogsteen type pairs are predominant in the A-U dimers.