Right-handed Helical Structure Research Articles

The pKa's of 2‘-Hydroxyl Group in Nucleosides and Nucleotides

This thesis is based on nine research publications (I – IX) on structure and reactivity of RNA vis-a-vis DNA. The DNA and RNA are made of flexible pentose sugar units, polyelectrolytic phosphodiester backbone, and heterocyclic nucleobases. DNA stores our genetic code, whereas RNA is involved both in protein biosynthesis and catalysis. Various ligand-binding and recognition properties of DNA/RNA are mediated through inter- and intra-molecular H-bonding and stacking interactions, beside hydration, van der Waal and London dispersion forces. In this work the pH dependant chemical shift, pKa values of 2'-OH group as well as those the nucleobases in different sequence context, alkaline hydrolysis of the internucleotidic phosphodiester bonds and analysis of NOESY footprints along with NMR constrained molecular dynamics simulation were used as tools to explore and understand the physico-chemical behavior of various nucleic acid sequences, and the forces involved in their self-assembly process. Papers I – II showed that the ionization of 2'-OH group is nucleobase-dependant. Paper III showed that the chemical characters of internucleotidic phosphate are non-identical in RNA compared to that of DNA. Papers IV – VI show that variable intramolecular electrostatic interactions between electronically coupled nearest neighbor nucleobases in a ssRNA can modulate their respective pseudoaromatic character, and result in creation of a unique set of aglycons with unique properties depending on propensity and geometry of nearest neighbor interaction. Paper VII showed that the cross-modulation of the pseudoaromatic character of nucleobases by the nearest neighbor is sequence-dependant in nature in oligonucleotides. Paper VIII showed that the purine-rich hexameric ssDNA and ssRNA retain the right-handed helical structure (B-type in ssDNA and A-type in ssRNA) in the single-stranded form even in absence of intermolecular hydrogen bonding. The directionality of stacking geometry however differs in ssDNA compared to ssRNA. In ssDNA the relatively electron-rich imidazole stacks above the electron-deficient pyrimidine in the 5' to 3' direction, in contradistinction, the pyrimidine stacks above the imidazole in the 5' to 3' direction in ssRNA. Paper IX showed that the pKa values of the nucleobases in monomeric nucleotides can be used to show that a RNA-RNA duplex is more stable than a DNA-DNA duplex. The dissection of the relative strength of base-pairing and stacking showed that the relative contribution of former compared to that of the latter in an RNA-RNA over the corresponding DNA-DNA duplexes decreases with the increasing content of A-T/U base pairs in a sequence.

NMR Characterization of a DNA Duplex Containing the Major Acrolein-derived Deoxyguanosine Adduct γ-OH-1,-N2-Propano-2′-deoxyguanosine

The environmental and endogenous mutagen acrolein reacts with cellular DNA to produce several isomeric 1,N(2)-propanodeoxyguanosine adducts. High resolution NMR spectroscopy was used to establish the structural features of the major acrolein-derived adduct, gamma-OH-1,N(2)-propano-2'-deoxyguanosine. In aqueous solution, this adduct was shown to assume a ring-closed form. In contrast, when gamma-OH-1,N(2)-propano-2'-deoxyguanosine pairs with dC at the center of an 11-mer oligodeoxynucleotide duplex, the exocyclic ring opens, enabling the modified base to participate in a standard Watson-Crick base pairing alignment. Analysis of the duplex spectra reveals a regular right-handed helical structure with all residues adopting an anti orientation around the glycosidic torsion angle and Watson-Crick alignments for all base pairs. We conclude from this study that formation of duplex DNA triggers the hydrolytic conversion of gamma-OH-1,N(2)-propano-2'-deoxyguanosine to an open chain form, a structure that facilitates pairing with dC during DNA replication and accounts for the surprising lack of mutagenicity associated with this DNA adduct.

A Chirally Stable, Atropoisomeric, C-Tetrasubstituted -Amino Acid: Incorporation into Model Peptides and Conformational Preference

A variety of model peptides, including four complete homologous series, to the pentamer level, characterized by the recently proposed binaphthyl-based, axially chiral, Cα-tetrasubstituted, cyclic α-amino acid Bin, in combination with Ala, Gly, or Aib residues, was synthesized by solution methods and fully characterized. The solution conformational propensity of these peptides was determined by FT-IR absorption and 1H-NMR techniques. Moreover, the molecular structures of the free amino acid (S)-enantiomer and an Nα-acylated dipeptide alkylamide with the heterochiral sequence -(R)-Bin-Phe- were assessed in the crystal state by X-ray diffraction. Taken together, the results point to the conclusion that β-bends and 310 helices are preferentially adopted by Bin-containing peptides, although the fully extended conformation would also be adopted in solution by the short oligomers to some extent. We also confirmed the tendency of (R)-Bin to fold a peptide chain into right-handed bend and helical structures. The absolute configuration of the Bin residue(s) was correlated with the typically intense exciton-split Cotton effect of the 1Bb binaphthyl transition near 225 nm.

A Chirally Stable, Atropoisomeric,Cα-Tetrasubstitutedα-Amino Acid: Incorporation into Model Peptides and Conformational Preference

A variety of model peptides, including four complete homologous series, to the pentamer level, characterized by the recently proposed binaphthyl-based, axially chiral, Cα-tetrasubstituted, cyclic α-amino acid Bin, in combination with Ala, Gly, or Aib residues, was synthesized by solution methods and fully characterized. The solution conformational propensity of these peptides was determined by FT-IR absorption and 1H-NMR techniques. Moreover, the molecular structures of the free amino acid (S)-enantiomer and an Nα-acylated dipeptide alkylamide with the heterochiral sequence -(R)-Bin-Phe- were assessed in the crystal state by X-ray diffraction. Taken together, the results point to the conclusion that β-bends and 310 helices are preferentially adopted by Bin-containing peptides, although the fully extended conformation would also be adopted in solution by the short oligomers to some extent. We also confirmed the tendency of (R)-Bin to fold a peptide chain into right-handed bend and helical structures. The absolute configuration of the Bin residue(s) was correlated with the typically intense exciton-split Cotton effect of the 1Bb binaphthyl transition near 225 nm.

Conformational Free Energy Surfaces of Ala10and Aib10Peptide Helices in Solution

A newly developed molecular simulation algorithm, the multidimensional conformational free energy thermodynamic integration (CFTI) method, has been applied to describe the conformational free energy surfaces of regular peptide helices in solution. The systems studied are (Ala)10 and (Aib)10 peptides, where Aib is α-methylalanine, in water and DMSO solution. The CFTI approach was used to calculate two-dimensional maps of the conformational free energy and its gradient as a function of the peptide backbone dihedrals (φ,ψ). In the region of right-handed helical structures of (Ala)10 and (Aib)10, the α-helix and π-helix were found to be locally stable states, corresponding to free energy minima. The location of the minima was refined by free energy optimization. Unexpectedly, solvation by both water and DMSO tended to strongly stabilize the π-helix relative to the standard α-helical structure. The π and α-helices had essentially identical stability for (Ala)10 in water and DMSO. The (Ala)10 π-helical free ene...

Assemblies of aziridinemethanols

Two novel long chain aziridinemethanols (1b, c) are described and their molecular organisation in the bulk and self-assembling properties in aqueous dispersion are reported. The orientation of the NH hydrogen of the aziridinealcohol moiety in 1b can be changed by introducing a methyl substituent into the rigid three-membered ring (1c), leading to a change in the hydrogen bonding pattern interconnecting these molecules. This change in configuration leads to marked differences in the ordering of these molecules in the solid state. Although compounds 1b and 1c both form highly organised structures in aqueous media and on the air–water interface, noteworthy differences are observed. Compound 1c yields left-handed helical ribbons whereas no chiral aggregates are found for 1b. However, the addition of 2-acetoxybenzoic acid (aspirin) to an aqueous dispersion of 1b leads to the generation of both left- and right-handed helical structures. Under these conditions a reaction had taken place that was specific for the ortho-isomer of acetoxybenzoic acid.

Helical superstructure from an achiral banana-shaped liquid crystal

In the banana-shaped achiral molecular system reported here, 1,3-phenylene bis[4-(3-chloro-4-n-octyloxy-phenyliminomethyl) benzoate] (PBCOB) and 1,3-phenylene bis[4-(3-fluoro-4-n-octyloxy-phenyliminomethyl) benzoate] (PBFOB), their smectic mesophases, including a switchable banana phase B7, were identified by differential scanning calorimetry (DSC), X-ray scattering and polarizing optical microscopy (POM) studies. Both the left- and right-handed helical domains are spontaneously formed upon cooling from isotropic liquid to the switchable banana phase (By). The homeotropic alignment and ferroelectric properties of banana-shaped liquid crystal are investigated. The spontaneous polarization for PBCOB is about 50 nC/cm2. Doping the PBCOB with a cyanohydrin-contain chiral smectic liquid crystal resulted in more right-handed helical structures.

Cryoatomic Force Microscopy of Filamentous Actin

Cryoatomic force microscopy (cryo-AFM) was used to image phalloidin-stabilized actin filaments adsorbed to mica. The single filaments are clearly shown to be right-handed helical structures with a periodicity of ∼38 nm. Even at a moderate concentration (∼10 μg/ml), narrow, branched rafts of actin filaments and larger aggregates have been observed. The resolution achieved is sufficient to resolve actin monomers within the filaments. A closer examination of the images shows that the branched rafts are composed of up to three individual filaments with a highly regular lateral registration with a fixed axial shift of ∼13 nm. The implications of these higher-order structures are discussed in terms of x-ray fiber diffraction and rheology of actin gels. The cryo-AFM images also indicate that the recently proposed model of left-handed F-actin is likely to be an artifact of preparation and/or low-resolution AFM imaging.

Solution structure of an 11-mer duplex containing the 3, N4-ethenocytosine adduct opposite 2′-deoxycytidine: implications for the recognition of exocyclic lesions by DNA glycosylases

Lipid peroxidation products, as well as the metabolic products of vinyl chloride, react with cellular DNA producing the mutagenic adduct 3, N 4-etheno-2′-deoxycytidine (ϵdC), along with several other exocyclic derivatives. High-resolution NMR spectroscopy and restrained molecular dynamics simulations were used to establish the solution structure of an 11-mer duplex containing an ϵdC·dC base-pair at its center. The NMR data suggested a regular right-handed helical structure having all residues in the anti orientation around the glycosydic torsion angle and Watson-Crick alignments for all canonical base-pairs of the duplex. Restrained molecular dynamics generated a three-dimensional model in excellent agreement with the spectroscopic data. The (ϵdC·dC)-duplex structure is a regular right-handed helix with a slight bend at the lesion site and no severe distortions of the sugar-phosphate backbone. The ϵdC adduct and its partner dC were displaced towards opposite grooves of the helix, resulting in a lesion-containing base-pair that was highly sheared but stabilized to some degree by the formation of a single hydrogen bond. Such a sheared base-pair alignment at the lesion site was previously observed for ϵdC·dG and ϵdC·T duplexes, and was also present in the crystal structures of duplexes containing dG·T and dG·U mismatches. These observations suggest the existence of a substrate structural motif that may be recognized by specific DNA glycosylases during the process of base excision repair.

The tetratricopeptide repeat: a structural motif mediating protein-protein interactions.

The tetratricopeptide repeat (TPR) motif is a protein-protein interaction module found in multiple copies in a number of functionally different proteins that facilitates specific interactions with a partner protein(s). Three-dimensional structural data have shown that a TPR motif contains two antiparallel alpha-helices such that tandem arrays of TPR motifs generate a right-handed helical structure with an amphipathic channel that might accommodate the complementary region of a target protein. Most TPR-containing proteins are associated with multiprotein complexes, and there is extensive evidence indicating that TPR motifs are important to the functioning of chaperone, cell-cycle, transcription, and protein transport complexes. The TPR motif may represent an ancient protein-protein interaction module that has been recruited by different proteins and adapted for specific functions. BioEssays 1999;21:932-939.

Synthesis and folding preferences of γ-amino acid oligopeptides: stereochemical control in the formation of a reverse turn and a helix

Abstract The stereoselective synthesis of α-cinnamyl γ-amino acids and the corresponding oligopeptides is described. Detailed 1D and 2D NMR studies in pyridine-d 5 show that the ( αR )-cinnamyl γ-amino acid tetrapeptide adopts a reverse turn structure, while the ( αS )-cinnamyl γ-amino acid tetrapeptide adopts a right-handed helical structure.

Self-assembly of disk-shaped molecules to coiled-coil aggregates with tunable helicity

A disk-shaped molecule with chiral tails is shown to form long fibers of molecular diameter and micrometer length by self-assembly in chloroform. The molecules are derived from crown ethers and contain a phthalocyanine ring. In the fibers, they have a clockwise, staggered orientation that leads to an overall right-handed helical structure. These structures, in turn, self-assemble to form coiled-coil aggregates with left-handed helicity. Addition of potassium ions to the fibers leaves their structure intact but blocks the transfer of the chirality from the tails to the cores, leading to loss of the helicity of the fibers. These tunable chiral materials have potential in optoelectronic applications and as components in sensor devices.

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.

Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine.

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.

Effect of Nα-Acyl Chain Length on the Membrane-Modifying Properties of Synthetic Analogs of the Lipopeptaibol Trichogin GA IV

Trichogin GA IV, an 11-residue lipopeptaibol blocked at the N-terminus by an n-octanoyl group and at the C-terminus by a 1,2-amino alcohol (l-leucinol), extracted from the fungus Trichoderma longibrachiatum, exhibits remarkable membrane-modifying properties. We have synthesized trichogin GA IV and several [l-Leu-OMe11] analogs carrying at the N-terminus an acyl chain of variable length (C2−C8, C10, C12, C14, C16, C18). A succinoylated head-to-head dimer was also prepared. A conformational analysis, carried out by FTIR absorption, CD, and NMR, showed that the right-handed helical structure of the natural lipopeptaibol is essentially preserved in all its analogs. Permeability measurements revealed that at least six carbon atoms in the Nα-blocking fatty acyl moiety are required for the onset of significant membrane-modifying properties. Also the head-to-head dimer is remarkably active. Possible models for the mechanism of membrane permeability of trichogin GA IV are discussed.

Isolation of intramitochondrial helical filaments appearing in outer compartment of mitochondria.

Packets of helical filaments have been observed in the outer compartment of occasional mitochondria in many cell types in a variety of animals. The composition and function of these intramitochondrial helical filaments (IMHF) are unknown. IMHF were induced in a hepatic mitochondria by administration of ethanol in the drinking water of rats. Hepatic mitochondria were isolated and ruptured by osmotic shock, releasing the IMHF. To purify these structures, the IMHF-containing supernatant was further fractionated by ammonium sulfate precipitation, a 50-60% solution of this reagent being the most effective in this regard. Isolated IMHF were examined by electron microscopy and were analyzed by SDS-PAGE. Isolated IMHF closely resembled their in situ counterparts: they had a right-handed helical structure with a 16 nm pitch. SDS-PAGE analysis revealed that they contained three polypeptides with molecular weight of 135, 98, and 53 kD, respectively. These observations will stand as a baseline for comparisons with IMHF that occur naturally or that are induced in other cell types by other kinds of experimental manipulation.

Effect of thymine tract length on the structure and stability of model telomeric sequences

DNA from the telomeres at the ends of eukaryotic chromosomes contains a stretch of simple tandemly repeated sequences in which clusters of G residues alternate with clusters of T/A sequences along one DNA strand. Model telomeric G-clusters form four-stranded structures in Na+ or K+, stabilized by Hoogsteen pairing between G bases. DNA containing a single copy of the G-cluster can self-associate to form tetramers, with a parallel-stranded, right-handed helical structure. Two copies of the 3'-terminal G strand form a folded-back hairpin that dimerizes to create an antiparallel quadruplex structure. We show here that the tetrameric structure is strongly influenced by the T residue flanking either side of the G-cluster. The parallel tetraplex formed by single copies of the sequences dTnG4 is most stable for n = 1 and least stable for n = 8, the longest tract we have studied. At least two thymine residues are required to allow formation of antiparallel folded-back hairpin dimers from two-copy oligomers of sequence d(TnG4)2 in Na+; additional T's destabilize this structure. In K+, the predominant structure formed is the four-stranded parallel tetramer in all cases. Kinetic analysis indicates that the quadruplex structure formed by Oxytricha telomeric DNA overhangs in the presence of Na+ arises by dimerization of two Hoogsteen base-paired hairpins, with a relatively low energy barrier.

Thermodynamics of G-tetraplex formation by telomeric DNAs.

Telomeres are structures at the ends of eukaryotic chromosomes, the DNA of which contains stretches of tandemly repeated sequences with G clusters along one strand. Model telomeric G-rich DNAs can form different tetraplex structures, stabilized by cyclic hydrogen bonding of four guanines in the presence of metal ions such as Na+ or K+. Oligonucleotides with a single copy of the Oxytricha sequence dT4G4 form a tetramer, with a parallel-stranded, right-handed helical structure. Additional copies favor folded-back structures that associate to form an antiparallel dimer. The parallel-stranded tetramer has all G's in the anti configuration, while the folded-back dimer has alternating syn and anti nucleotide conformations along each strand. Here we have constructed two G-tetraplex structures, containing identical G-tetrad base pairs, from oligonucleotides. One has the truncated telomeric sequence from Oxytricha, dG4T4G4, which forms an antiparallel G-quartet structure; the second is constrained to form a parallel G-strand arrangement by insertion of a 5'-p-5' linkage between two dT2G4 sequences. Each oligomer forms a defined G-tetraplex dimeric structure in the presence of Na+. The standard-state enthalpies, entropies, and free energy for formation of these tetraplexes have been determined. The parallel strand structure is thermodynamically more stable than the antiparallel one, primarily because of both greater enthalpy and entropy of formation. In addition, the two molecules differ in their interaction with sodium ions, reflecting a difference in ion binding and therefore in structure between the two forms.

Peptides from chiral Cα,α‐disubstituted glycines Synthesis and characterization, conformational energy computations and solution conformational analysis of Cα‐methyl, Cα‐isopropylglycine [(αMe)Val] derivatives and model peptides*

Conformational energy computations on Ac-L-(alpha Me)Val-NHMe indicate that turns and right-handed helical structures are particularly stable conformations for this chiral C alpha-methyl, C alpha-alkylglycyl residue. We have synthesized and characterized a variety of L-(alpha Me)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, 1H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the L-(alpha Me)Val residue turns out to be a strong beta-turn and right-handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other C alpha-methyl, C alpha-alkylglycyl residues.

Scanning tunneling microscopy of collagen molecules

Calf skin collagen molecules adsorbed on a graphite substrate were directly imaged by scanning tunneling microscopy in air. The right-handed helical structures were observed and the average periodicity was quantitatively measured to be 31 Å, in good agreement with the value of about 28 Å estimated from biochemical studies. The intermolecular distance in a collagen bundle was 23 Å.