Adjacent Strands Research Articles

Solution Structure of a DNA Quadruplex Containing the Fragile X Syndrome Triplet Repeat

Both X-ray and NMR structural studies have defined the polymorphic nature of G-quadruplexes generated through mutual stacking of G·G·G·G tetrads by guanine rich telomeric sequences. Recently, the fragile X syn- drome d(C-G-G) n triplet nucleotide repeat has been shown to form a stable quadruplex of undefined structure in monovalent cation solution. We have undertaken a structural characterization of the d(G-C-G-G-T 3-G-C-G-G) undecanucleotide to elucidate the structural alignments associated with quadruplex formation by this oligomer which contains sequence elements associated with the fragile X syndrome triplet repeat. d(G-C-G-G-T 3-G-C- G-G) in Na +cation solution forms a quadruplex through dimerization of two symmetry related hairpins with the lateral connecting T 3loops positioned at opposite ends of the quadruplex. This novel NMR-molecular dynamics based solution structure contains internal G·C·G·C tetrads sandwiched between terminal G·G·G·G tetrads. Watson-Crick G·C base-pairs within individual hairpins dimerize through their major groove edges using bifurcated hydrogen bonds to form internal G( anti)·C( anti)·G( anti)·C( anti) tetrads. Adjacent strands are anti-parallel to each other around the symmetric G-quadruplex which contains two distinct narrow and two symmetric wide grooves. By contrast, the terminal G-tetrads adopt G( syn)·G( anti)·G( syn)·G( anti) alignments. The structure of the d(G-C-G-G-T 3-G-C-G-G) quadruplex with its multi-layer arrangement of G·G·G·G and G·C·G·C tetrads greatly expands on our current knowledge of quadruplex folding topologies. Our results establish the pairing alignments that can be potentially utilized by the fragile X syndrome triplet repeat to form quadruplex structures through dimerization of hairpin stems. The formation of novel G·C·G·C tetrads through dimerization of Watson-Crick G·C base-pairs is directly relevant to the potential pairing alignments of helical stems in genetic recombination.

The Occurence of C–H · · · O Hydrogen Bonds in Proteins

Hydrogen bonds are a major feature of protein structure. By a generally accepted definition, they occur whenever a proton is shared by two electronegative atoms. Hence, only hydrogens bonded to nitrogen and oxygen atoms are usually considered in analysis of protein hydrogen bond networks. However, X-ray and neutron diffraction studies have shown that crystals of various organic compounds exhibit close C–H · · · X contacts (where X is an electronegative atom, in most cases oxygen) which show all the stereochemical hallmarks of hydrogen bonds. In this work, we describe an analysis of short C–H · · · O interactions in a sample of known protein structures representing different categories of tertiary folds and refined at a resolution of at least 2 Å. Although our analysis is based on the calculated coordinates of hydrogen atoms, its results are statistically significant: we find strong evidence that a large percentage of short C· · · O contacts constitute cohesive interactions. Moreover, the stereochemical study of C–H · · ·O=C contacts, in which the orientation of free electron orbitals on the acceptor oxygen atom can be predicted, reveals that these interactions exhibit stereochemical feature typical of hydrogen bonds. Among the hydrogen atoms involved in these contacts, the most common are those bonded to α carbon. This is consistent with the fact that these hydrogens are more acidic than others. We describe four different categories of C–H · · · O=C bonds. Those found between C α–H groups and main chain oxygens in adjacent strands of β sheets are the most ubiquitous. Our results call for a revision of crystallographic restrained refinement programs which treat close carbon–oxygen contacts as purely repulsive; they may also have implications for the understanding of some enzymatic reaction mechanisms.

An RBD that does not bind RNA: NMR secondary structure determination and biochemical properties of the C-terminal RNA binding domain from the human U1A protein.

We have obtained backbone 1H, 15N, and 13C assignments and determined the secondary structure and folding topology of the C-terminal RNA-binding domain (RBD) of the human U1A protein. The secondary structure derived from NOE data is in excellent agreement with the predicted structure from the 1H and 13C chemical shift indices. This 88 amino acid domain exhibits a beta alpha beta-beta alpha beta folding pattern, with conserved RNP1 and RNP2 sequences located in two adjacent strands of a four-strand antiparallel beta-sheet. This global folding pattern is typical of this class of RNA binding proteins. Although this domain contains residues that are conserved in all RBDs, its RNA binding properties are very unusual. RNA binding studies show that this domain does not bind U1, U2 or U5 snRNA, an RNA hairpin, rA16, rU16, rC16 or rA3U3GUA4, nor does it show significant association to populations of random sequence RNAs.

An analytical method for plain weave fabric composites

A two-dimensional closed-form analytical method has been developed for the thermoelastic analysis of two-dimensional orthogonal plain weave fabric laminae. Strand undulation and continuity in both the warp and fill directions, actual strand cross-section and weave geometry, strand fibre volume fraction and possible gap between the two adjacent strands have been considered in the analysis. Good correlation is observed between the predicted thermoelastic properties and experimental results.

An RBD that does not bind RNA: NMR secondary structure determination and biochemical properties of the C-terminal RNA binding domain from the human U1A protein

We have obtained backbone 1H, 15N, and 13C assignments and determined the secondary structure and folding topology of the C-terminal RNA-binding domain (RBD) of the human U1A protein. The secondary structure derived from NOE data is in excellent agreement with the predicted structure from the 1H and 13C chemical shift indices. This 88 amino acid domain exhibits a beta alpha beta-beta alpha beta folding pattern, with conserved RNP1 and RNP2 sequences located in two adjacent strands of a four-strand antiparallel beta-sheet. This global folding pattern is typical of this class of RNA binding proteins. Although this domain contains residues that are conserved in all RBDs, its RNA binding properties are very unusual. RNA binding studies show that this domain does not bind U1, U2 or U5 snRNA, an RNA hairpin, rA16, rU16, rC16 or rA3U3GUA4, nor does it show significant association to populations of random sequence RNAs.

Identification by in Vitro Mutagenesis of the Interaction of Two Segments of C2MstC1, a Chimera of Cytochromes P450 2C2 and P450 2C1

A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450cam, P450BM-3, and P450terp predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.

Three-dimensional structure of a lipoyl domain fromthe dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The structure of a lipoyl domain from the pyruvate dehydrogenasemultienzyme complex of Escherichia coli has been determined by means of nuclear magnetic resonance spectroscopy. A total of 549 nuclear Overhauser effect distance restraints, 52torsion angle restraints and 16 slowly exchanging amide protons were employed as input for the structure calculations. These were performed using a combined distance geometry-simulated annealing strategy. The domain is a hybrid between the N and C-terminal halves of the first and third lipoyl domains, respectively, of the dihydrolipoyl acetyltransferase component of the E. coli multienzyme complex, representing residues 1 to 33 and 238 to 289 (wild-type numbering). The lipoyl-lysine residue was also replaced by glutamine. Nonetheless, its structure, two four-stranded β-sheets forming a flattened β-barrel, closely resembles that of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilits determined previously. As before, the lipoylation site is physically exposed in a tight turn in one of the β-sheets, and the N and C-terminal residues are close together at the other end of the molecule in adjacent strands of the other β-sheet. Another prominently conserved feature of the structure is the 2-fold axis of quasi-symmetry relating the N and C-terminal halves of the domain. Consistent with the high level of sequence similarity between lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes from many different sources, these results confirm that all lipoyl domains are likely to have closely related structures.

Conformational polymorphism in telomeric structures: Loop orientation and interloop pairing in d(G4TnG4)

Sequence repeats constituting the telomeric regions of chromosomes are known to adopt a variety of unusual structures, consisting of a G tetraplex stem and short stretches of thymines or thymines and adenines forming loops over the stem. Detailed model building and molecular mechanics studies have been carried out for these telomeric sequences to elucidate different types of loop orientations and possible conformations of thymines in the loop. The model building studies indicate that a minimum of two thymines have to be interspersed between guanine stretches to form folded-back structures with loops across adjacent strands in a G tetraplex (both over the small as well as large groove), while the minimum number of thymines required to build a loop across the diagonal strands in a G tetraplex is three. For two repeat sequences, these hairpins, resulting from different types of folding, can dimerize in three distinct ways--i.e., with loops across adjacent strands and on same side, with loops across adjacent strands and on opposite sides, and with loops across diagonal strands and on opposite sides--to form hairpin dimer structures. Energy minimization studies indicate that all possible hairpin dimers have very similar total energy values, though different structures are stabilized by different types of interactions. When the two loops are on the same side, in the hairpin dimer structures of d(G4TnG4), the thymines form favorably stacked tetrads in the loop region and there is interloop hydrogen bonding involving two hydrogen bonds for each thymine-thymine pair. Our molecular mechanics calculations on various folded-back as well as parallel tetraplex structures of these telomeric sequences provide a theoretical rationale for the experimentally observed feature that the presence of intervening thymine stretches stabilizes folded-back structures, while isolated stretches of guanines adopt a parallel tetraplex structure.

Wear-Related Fatigue in a Wire Rope Failure

Abstract The fatigue failure of a nonrotating wire rope used on a skip hoist in an underground mine has been studied as part of the ongoing research by the U.S. Bureau of Mines into haulage and materials handling hazards in mines. Correlation of individual wire failures within two segments of the failed rope were used to gain an understanding of the progression of degradation leading to rope failure. Wire failures occurred predominantly at locations of wear between adjacent strands. These interstrand wear sites are identifiable by a large reduction in diameter; however, reduction in area is not responsible for the location of failure. Fractography revealed crack initiation sites to be located opposite the characteristic wear site or at other less noticeable wear sites. The primary failure mechanism of individual wires within the rope is believed to be a function of contact stress at the interstrand wear sites. Axially loading and unloading the rope produces high compressive stresses at the contact sites, which in turn produce large alternating tensile stresses on the opposite side of the wire. This mechanism has been termed “contact stress-accelerated fatigue.” This mechanism and the affiliated wear pattern both are consequences of interstrand contact.

Structure of ribonucleotide reductase protein R1.

Ribonucleotide reductase is the only enzyme that catalyses de novo formation of deoxyribonucleotides and is thus a key enzyme in DNA synthesis. The radical-based reaction involves five cysteins. Two redox-active cysteines are located at adjacent antiparallel strands in a new type of ten-stranded alpha/beta-barrel, and two others at the carboxyl end in a flexible arm. The fifth cysteine, in a loop in the centre of the barrel, is positioned to initiate the radical reaction.

The Footprint of Chromosomal Proteins HMG-14 and HMG-17 on Chromatin Subunits

The position of chromosomal proteins HMG-14 and HMG-17 in nucleosome cores and in chromatosomes lacking linker histones has been mapped by hydroxyl radical footprinting. Both the nucleosome core and the H1/H5 depleted chromatosome can specifically bind two molecules of HMG-14/-17. The path of HMG-14 on the surface of chromatin subunits is indistinguishable from that of HMG-17. The bound HMGs protect the DNA from hydroxyl radical cleavage 25 base-pairs from the end of the DNA in nucleosome cores in each of the two major grooves of the DNA flanking the nucleosomal dyad axis. Thus, in both cores and H1/H5-depleted chromatosomes the proteins bridge two adjacent DNA strands on the surface of the particles. The sites occupied by HMG near the end of the chromatosome-length particles are distinct from those occupied by the H1/H5 linker histones. In the region of the dyad axis the binding sites of HMGs overlap those of the linker histones. The placement of HMG-14/17 near the nucleosomal dyad axis raises the possibility that interactions between histone H1 and HMGs may affect the transcriptional potential of chromatin.

Strand orientation in the DNA quadruplex formed from the Oxytricha telomere repeat oligonucleotide d(G4T4G4) in solution.

The structure formed from the DNA oligonucleotide d(G4T4G4) (Oxy-1.5), which contains the Oxytricha telomere repeat T4G4, has been investigated by two-dimensional 1H and 31P NMR spectroscopy. Sequence-specific assignments have been obtained for the 1H and 31P resonances, using a combination of methods including comparisons to the inosine- and uracil-containing derivatives d(G4T4G3I) and d(G4UT3G4). The oligonucleotide forms a symmetrical bimolecular G-quadruplex with four G-quartets and thymine loops at opposite ends of the G-quartets. Guanines are alternatively syn and anti along each "strand" and all of the thymines are anti. The thymines loop diagonally across the G-quartet, resulting in a structure in which adjacent strands are alternately parallel and antiparallel and the glycosidic torsion angles are syn-syn-anti-anti around each G-quartet. There are three different types of grooves, a wide, a narrow, and two medium grooves. A diagonally looped quadruplex is formed in the presence of both Na+ and K+ counterions. The model structure of Oxy-1.5 is compared to the recently published crystal structure of Oxy-1.5 (Kang et al., 1992), which contains many of the same features as those found in solution but differs in that the thymines loop across an edge of the G-quartet.

Functional interactions between K+ pore residues located in different subunits

The aqueous pore (P-region) of homotetrameric voltage-gated K+ channels has been modeled as a radially symmetrical eight-stranded antiparallel beta-barrel to which each of the four subunits contributes equally. This model has hydrogen bonding between residues located on adjacent subunits and predicts that subunit interactions might have functional consequences. Previously we have used point mutations and an electrophysiological assay to detect functional interactions between a pair of residues at positions 369 and 374 in the P-region, but we could not distinguish between intra- and intersubunit interactions. In the present paper, we present evidence for interaction across subunit boundaries after co-injecting two cRNAs encoding subunits differing from each other at either position 369 or 374. Comparison of the phenotypes of homo- and heterotetrameric channels suggests that pore residues residing in adjacent subunits form a closely packed structure which determines both ion conductance and stability of the open state of the channel. Our results are consistent with a structure in which pore residues 369 and 374 are located in close proximity on adjacent antiparallel strands to allow both intra- and intersubunit interactions.

Peptide spontaneously assembles into membrane

A 16-residue peptide has been found to spontaneously assemble into a visible, insoluble membrane—a phenomenon that could have implications for neurological, biomaterials, and origin-of-life studies. The discovery was made by biologists Shuguang Zhang, Todd Holmes, Curtis Lockshin, and Alexander Rich at Massachusetts Institute of Technology [ Proc. Natl. Acad. Sci. USA , 90, 3334 (1993)]. We've done a lot of literature searching, and it seems to be the first peptide that self-assembles into a macroscopic membrane, says Rich. The peptide, which the researchers call EAK16, consists of alternating hydrophobic residues (alanine) and hydrophilic residues (glutamine and lysine). The sequence was found in a natural yeast protein that was originally identified by the researchers for its ability to bind left-handed Z-DNA. The hydrophilic residues are of opposite charge, giving adjacent peptide strands ionic complementarity. The peptide is soluble in water, but upon addition of salt it comes out of solution and ...

Cross-reactive T cell clones from unrelated individuals reveal similarities in peptide presentation between HLA-B27 and HLA-DR2.

HLA-B27- responder cells were stimulated in vitro with B*2705+ lymphoblastoid cell lines and alloreactive CTL clones were obtained by limiting dilution. Of the CD3+ CD4-CD8+ HLA-B27-specific CTL clones obtained, two of them, possessing the same TCR, cross-reacted with HLA-DR2. The fine specificity of these CTL was established with HLA-B27 and HLA-DR2 subtypes. They recognized the B*2701 to B*2706 subtypes, but only DR2Dw2. Lysis of DR2+ target cells was specifically inhibited by anti-CD3, anti-class II, and anti-DR mAb, but not with an anti-CD8 antibody. The monoclonal nature of the cross-reaction was established by the mutual inhibition of HLA-B27 and DR2Dw2 cells in cold target competition experiments. The DR2 protein involved in the cross-reaction was the heterodimer carrying the B5*0101 product, as shown by using L cell transfectants expressing each of the two molecules encoded in the DR2Dw2 haplotype. A correlation between the fine specificity of these CTL clones and the amino acid sequences of HLA-B27 and HLA-DR2 subtypes revealed a shared structural motif between HLA-B27 and the DR2 B5*0101 chain, which could be related to the observed cross-reaction. This motif was contributed for by several residues located in adjacent beta strands, at the floor of the peptide-binding site. The contribution of two of these residues, as well as other beta-pleated sheet residues to HLA-B27 allorecognition by the cross-reactive CTL clones was directly demonstrated with site-directed mutants. These results suggest that the dual reactivity pattern reflects presentation of identical or structurally related peptide by HLA-B27 and HLA-DR2Dw2. As T cell cross-reactivity between HLA-B27 and HLA-DR2 was previously found in cells from an unrelated individual the results reported here are likely to reflect an intrinsic property of HLA-B27, rather than the fortuitous finding of a rare clonal reaction pattern. We speculate on the potential implications of these results for the pathogeny of HLA-B27-associated spondyloarthropathies.

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.

Prediction of thermal expansion coefficients of plain weave fabric composites

Three plain weave fabric composite analysis models are presented for the prediction of the on-axes thermal expansion coefficients. These are two-dimensional models in the sense that the actual strand cross-sectional geometry, strand undulation and the presence of a gap between the adjacent strands are taken into account. In the first two models, termed refined models, the representative unit cell is discretised into slices and elements and analysed. In the third method, a closed-form solution is presented. In this case, the representative unit cell is idealised as a crossply laminate and analysed. The relative merits and demerits of the models are also discussed. The predicted results are compared with the experimental values. A good correlation is observed.

Proteoglycans from the swarm rat chondrosarcoma. Structure of the aggregates extracted with associative and dissociative solvents as revealed by electron microscopy.

Proteoglycan aggregates were extracted from Swarm rat chondrosarcoma tissue in the native state and compared with proteoglycan aggregates isolated dissociatively with 4 M guanidine HCl. Purified aggregates were examined with a variety of electron microscopic techniques. In some cases they showed a structure of the central filament identical to that of the link-stabilized central filament observed in earlier experiments where the separated constituents were allowed to reconstitute (Mörgelin, M., Paulsson, M., Hardingham, T. E., Heinegård, D., and Engel, J. (1988) Biochem. J. 253, 175-185). The tight packing of proteoglycan monomers along the hyaluronate with a minimum distance of 12 nm between adjacent E1 strands also could thus be confirmed for never dissociated aggregates. The results therefore show that the organization of proteoglycan aggregates assembled in vitro from the participating molecules is representative for conditions in situ. An additional structural type of central filament was observed in the preparations. This contained long stretches of free hyaluronate interspaced by short stretches of central filament with condensed arrays of link protein-proteoglycan. Chemical cross-linking in combination with low shear electron microscopical techniques showed that this discontinuous central filament structure is not an artifact of specimen preparation. The addition of suprastoichiometric amounts of exogenous link protein did not affect the central filament structure with the low packing density. Densely and loosely packed types of central filament were isolated in varying relative amounts with different associative and dissociative solvents.

Quadruplex structure of Oxytricha telomeric DNA oligonucleotides.

The telomeres of most eukaryotes contain a repeating G-rich sequence with the consensus d(T/A)1-4G1-8, of which 12-16 bases form a 3' single-strand overhang beyond the telomeric duplex. It has been proposed that these G-rich oligonucleotides associate to form four-stranded structures from one, two or four individual strands and that these structures may be relevant in vivo. The proposed structures contain Hoogsteen base-paired G-quartets, precedent for which has been in the literature for many years. Here we use 1H NMR spectroscopy to study the conformations of the DNA oligonucleotides d(G4T4G4) (Oxy-1.5) and d(G4T4G4T4G4T4G4) (Oxy-3.5) which contain the Oxytricha telomere repeat (T4G4). We find that these molecules fold to form a symmetrical bimolecular and an intramolecular quadruplex, respectively. Both structures have four G-quartets formed from nucleotides that are alternately syn and anti along each strand. This arrangement differs from earlier models in which the strands are alternately all syn or all anti. The T4 loops in Oxy-1.5 are on opposite ends of the quadruplex and loop diagonally across the G-quartet, resulting in adjacent strands being alternately parallel and antiparallel.

Stereochemical complementarity of progesterone, RU486 and cavities between base pairs in partially unwound double stranded DNA assessed by computer modeling and energy calculations

Computer modeling was used to examine the relative fit of progesterone and RU486 in cavities constructed between base pairs in double stranded DNA. Progesterone was capable of forming two stereospecific hydrogen bonds between the carbonyl groups and protonated phosphate groups on adjacent strands. Favorable van der Waals and electrostatic energies were exhibited upon insertion of progesterone into DNA indicating an excellent fit. While RU486 could be accommodated between the base pairs and formed hydrogen bonds, there was a high van der Waals energy in the resulting complex. When the complexes were subjected to energy minimization, the conformation of the DNA was significantly altered in the RU486-DNA complex but not in the progesterone-DNA complex. No mechanistic interpretation of these results is proffered; however, such information may have evolutionary significance and could prove useful in designing new progesterone agonists and antagonists.