A study of the B-Z transition of the AC-rich region of the repeat unit of a satellite DNA from Cebus by means of chemical probes

The conformational changes induced by negative supercoiling in the AC-rich region of the repeat unit of a Cebus satellite DNA has been studied by chemical probes sensitive to alterations in DNA conformation. This region is constituted of a (GT/CA)n stretch (15 less than or equal to n less than or equal to 18) associated to a sequence rich in GT/CA. At high superhelical density, at least 100 base pairs in the AC-rich region adopt the Z conformation as judged by diethyl pyrocarbonate reactivity. This is confirmed by diethyl pyrocarbonate footprinting of the complex between antibodies to Z-DNA and the AC-rich region. Osmium tetroxide and hydroxylamine reveal some distortions of the Z double helix in the (GT/CA)n stretch also. The terminal T residues of the stretch are hyperreactive with osmium tetroxide; the terminal left C residues but not the terminal right C residues are hyperreactive with hydroxylamine. Substitution of a few base pairs in the middle of the (GT/CA)n stretch induces also some distortions of the Z double helix. In the GT/CA-rich sequence, distortion of the Z double helix is also supported by the hyperreactivity of osmium tetroxide with several T and C residues.     

The conformation and stability of ribonucleic acids: modeling base sequence effects in double stranded helices

Base sequence effects within double stranded RNA oligomers of A and Z conformations have been studied by molecular modeling using a methodological approach specifically adapted to nucleic acids. Calculations on symmetric oligomers having homonucleotide or dinucleotide repeating base sequences show that sequence changes can produce modifications in overall conformation, influence the degree of internal hydrogen bonding and strongly affect stability.     

An investigation of the conformational properties of ribosomes using N-ethylmaleimide as a probe.

The reactivity of ribosomal proteins towards N-ethylmaleimide has been examined in a variety of ribosome and ribosomal subunit preparations from Escherichia coli. The data show that samples which would be regarded as equivalent operationally can differ significantly in conformation, as judged by reactivity, depending on the method of preparation. The washing of ribosomes with high concentrations of salt has a particularly dramatic effect on protein reactivity. The implications of these results for our understanding of ribosome conformation and for the further study of conformation by chemical reactivity are discussed.     

Importance of conserved residues for the conformation of the T-loop in tRNAs

The conformation of the T-loop of yeast tRNA(Asp) was studied by structural mapping techniques using chemical and enzymatic probes and by three-dimensional graphics modeling with the known crystallographic structures of tRNAs as references. The structural importance of C61 (conserved in the T-stem of all tRNAs) for the loop conformation was directly checked by ethylnitrosourea phosphate alkylation, either on the 3'-half tRNAAsp molecule or on a variant in which C61 was replaced by U61. The reactivity of P60 against ethylnitrosourea alkylation in the variant emphasizes the role of the hydrogen bond between this phosphate and position N4 of C61 for stabilizing the conformation of the T-loop. Experiments on several tRNA variants, containing C61 but altered in the sequence or in the length of the T-loop, indicate that other structural features help to stabilize the hydrogen bond network around P60. Evidence is presented that the reverse Hoogsteen base pair T54-A58 contributes to this stabilization by maintaining the hydrogen bonding between the 2'OH of ribose 58 and P60. Using graphics modeling and based on the chemical data. T-loops of several variants were constructed. It appears that both the constant length of the T-loop and the presence of psi 55 are crucial for the correct interaction between the T- and D-loops. The conclusion of this study is that the T-loop in tRNA possesses an intrinsic conformation (mainly governed by the constant residues) existing primarily without the structural context of the entire tRNA molecule.     

(A-T)n tracts embedded in random sequence DNA--formation of a structure which is chemically reactive and torsionally deformable.

Alternating d(A-T)n sequences which are contiguous with DNA of effectively random sequence have an abnormal conformation in linear DNA molecules. These regions are strongly reactive towards chemical modification by osmium tetroxide, and are preferentially cleaved by micrococcal nuclease. Both the chemical modification and the enzymic cutting occur uniformly through the alternating tract, and there is no evidence for enzyme or chemical sensitivity in the interfaces between the tract and DNA of normal conformation. These reactivities have a requirement for an alternating sequence. In addition to chemical reactivity, alternating (A-T)n sequences exhibit anomalously small twist changes on cruciform formation, suggesting that the pre-extruded DNA is underwound. We propose that the alternating sequences adopt an altered conformation which is subject to easy torsional deformation.     

Mapping the binding site of aflatoxin B1 in DNA: systematic analysis of the reactivity of aflatoxin B1 with guanines in different DNA sequences

The mutagenic and carcinogenic chemical aflatoxin B1 (AFB1) reacts almost exclusively at the N(7)-position of guanine following activation to its reactive form, the 8,9-epoxide (AFB1 oxide). In general N(7)-guanine adducts yield DNA strand breaks when heated in base, a property that serves as the basis for the Maxam-Gilbert DNA sequencing reaction specific for guanine. Using DNA sequencing methods, other workers have shown that AFB1 oxide gives strand breaks at positions of guanines; however, the guanine bands varied in intensity. This phenomenon has been used to infer that AFB1 oxide prefers to react with guanines in some sequence contexts more than in others and has been referred to as "sequence specificity of binding". Herein, data on the reaction of AFB1 oxide with several synthetic DNA polymers with different sequences are presented, and (following hydrolysis) adduct levels are determined by high-pressure liquid chromatography. These results reveal that for AFB1 oxide (1) the N(7)-guanine adduct is the major adduct found in all of the DNA polymers, (2) adduct levels vary in different sequences, and, thus, sequence specificity is also observed by this more direct method, and (3) the intensity of bands in DNA sequencing gels is likely to reflect adduct levels formed at the N(7)-position of guanine. Knowing this, a reinvestigation of the reactivity of guanines in different DNA sequences using DNA sequencing methods was undertaken. The reactivities of 190 guanines were determined quantitatively and considered in a pentanucleotide context, 5'-WXGYZ-3', where the central, underlined G represents the reactive guanine and W, X, Y, and Z can be any of the nucleotide bases. Methods are developed to determine that the X (5'-side) base and the Y (3'-side) base are most influential in determining guanine reactivity. The influence of the bases in the 5'-position (X) is 5'-G (1.0) greater than C (0.8) greater than A (0.3) greater than T (0.2), while the influence of the bases in the 3'-position (Y) is 3'-G (1.0) greater than T (0.8) greater than C (0.4) greater than A (0.3). These rules in conjunction with molecular modeling studies (to be published elsewhere) were used to assess the binding sites that might be utilized by AFB1 oxide in its reaction with DNA.     

Preferential binding and structural distortion by Fe2+ at RGGG-containing DNA sequences correlates with enhanced oxidative cleavage at such sequences

Certain DNA sequences are known to be unusually sensitive to nicking via the Fe2+-mediated Fenton reaction. Most notable are a purine nucleotide followed by three or more G residues, RGGG, and purine nucleotides flanking a TG combination, RTGR. Our laboratory previously demonstrated that nicking in the RGGG sequences occurs preferentially 5' to a G residue with the nicking probability decreasing from the 5' to 3'end of these sequences. Using 1H NMR to characterize Fe2+ binding within the duplex CGAGTTAGGGTAGC/GCTACCCTAACTCG and 7-deazaguanine-containing (Z) variants of it, we show that Fe2+ binds preferentially at the GGG sequence, most strongly towards its 5' end. Substitutions of individual guanines with Z indicate that the high affinity Fe2+ binding at AGGG involves two adjacent guanine N7 moieties. Binding is accompanied by large changes in specific imino, aromatic and methyl proton chemical shifts, indicating that a locally distorted structure forms at the binding site that affects the conformation of the two base pairs 3' to the GGG sequence. The binding of Fe2+ to RGGG contrasts with that previously observed for the RTGR sequence, which binds Fe2+ with negligible structural rearrangements.     

Interaction of collagen-like peptide models of asymmetric acetylcholinesterase with glycosaminoglycans: spectroscopic studies of conformational changes and stability

The effect of heparin on the conformation and stability of triple-helical peptide models of the collagen tail of asymmetric acetylcholinesterase expands our understanding of heparin interactions with proteins and presents an opportunity for clarifying the nature of binding of ligands to collagen triple-helix domains. Within the collagen tail of AChE, there are two consensus sequences for heparin binding of the form BBXB, surrounded by additional basic residues. Circular dichroism studies were used to determine the effect of the addition of increasing concentrations of heparin on triple-helical peptide models for the heparin binding domains, including peptides in which the basic residues within and surrounding the consensus sequence were replaced by alanine residues. The addition of heparin caused an increased triple-helix content with saturation properties for the peptide modeling the C-terminal site, while precipitation, with no increased helix content resulted from heparin addition to the peptide modeling the N-terminal site. The results suggest that the two binding sites with a similar triple-helical conformation have distinctive ways of interacting with heparin, which must relate to small differences in the consensus sequence (GRKGR vs GKRGK) and in the surrounding basic residues. Addition of heparin increased the thermal stability of all peptides containing the consensus sequence. Heparan sulfate produced conformational and stabilization effects similar to those of heparin, while chondroitin sulfate led to a cloudy solution, loss of circular dichroism signal, and a smaller increase in thermal stability. Thus, specificity in both the sequence of the triple helix and the type of glycosaminoglycan is required for this interaction.     

Theoretical studies of the electrostatic stability of A,B, C,and Z-DNA conformers as a function of monovalent ion concentration and ion type

Theoretical calculations using a two-state model of counterion phosphate interactions have been performed in order to determine the electrostatic stability of DNA in various ionic environments. Comparison of the stabilities of different conformations leads to results consistent with the B→Z transitions observed in high-salt environments and suggest that at very low ionic strnegths the left-handed Z(II) form is an electrostatically preferred conformation. An ion-type dependence of the high-salt B→Z transition midpoint is predicted. Calculations of the energies of the A and C conformations are also discussed in relation to experimentally observed transitions.     

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.     

6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.

Conformational diseases such as amyloidosis, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. These diseases are typified by the Z variant of alpha(1)-antitrypsin (E342K), which causes the retention of protein within hepatocytes as inclusion bodies that are associated with neonatal hepatitis and cirrhosis. The inclusion bodies result from the Z mutation perturbing the conformation of the protein, which facilitates a sequential interaction between the reactive center loop of one molecule and beta-sheet A of a second. Therapies to prevent liver disease must block this reactive loop-beta-sheet polymerization without interfering with other proteins of similar tertiary structure. We have used reactive loop peptides to explore the differences between the pathogenic Z and normal M alpha(1)-antitrypsin. The results show that the reactive loop is likely to be partially inserted into beta-sheet A in Z alpha(1)-antitrypsin. This conformational difference from M alpha(1)-antitrypsin was exploited with a 6-mer reactive loop peptide (FLEAIG) that selectively and stably bound Z alpha(1)-antitrypsin. The importance of this finding is that the peptide prevented the polymerization of Z alpha(1)-antitrypsin and did not significantly anneal to other proteins (such as antithrombin, alpha(1)-antichymotrypsin, and plasminogen activator inhibitor-1) with a similar tertiary structure. These findings provide a lead compound for the development of small molecule inhibitors that can be used to treat patients with Z alpha(1)-antitrypsin deficiency. Furthermore they demonstrate how a conformational disease process can be selectively inhibited with a small peptide.     

Tandem repeats of a specific alternating purine-pyrimidine DNA sequence adjacent to protamine genes in the rainbow trout that can exist in the Z form

We have located an extensive (AC)n-rich but specific sequence downstream of three rainbow trout protamine genes. Although sharing considerable sequence homology, including a perfectly conserved 46 base pair repeat, the sequences exhibit a regular heterogeneity in the length of the (AC)n-rich tracts. Radioimmunoassay experiments, S1 nuclease sensitivity studies, two-dimensional electrophoretic analysis, and immunoelectron microscopy studies have been used to determine if the region could assume a Z DNA conformation. It was found that, in a supercoiled plasmid, the (AC)n-rich region has the ability to attain the Z DNA conformation under physiological conditions.     

An examination of the binding behavior of histidine-containing peptides with immobilized metal complexes derived from the macrocyclic ligand, 1,4,7-triazacyclononane

In this study, two different experimental approaches have been employed to examine the binding behavior of histidine-containing peptides with metal ion complexes derived from the macrocyclic ligand 1,4,7-triazacyclononane (tacn). Firstly, a molecular modeling approach has been employed to derive the strain energies for test peptide sequences that have a predicted propensity to readily adopt an α-helical conformation. To this end, binuclear metal complexes were examined with peptides containing two histidine residues in different locations in a pair of peptides of the same composition but different sequence. These modeling results indicate that there are no energetic constraints for two-point binding to occur with dicopper(II) binuclear complexes when two histidine residues are appropriately placed in an α-helical conformation. Secondly, binding experiments were carried out to establish the effect of one or more histidine residues within a peptide sequence on the affinity of a peptide for these Cu(II)–tacn derived binuclear complexes when immobilized onto a chromatographic support material. The results confirm that for all chelating systems, higher affinity is achieved as the histidine number in the peptide structure increases, although the relative location of the histidine residues in these small peptides did not introduce a significant constraint to the conformation on interacting with the immobilized Cu(II) binuclear complexes.     

Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues (Mr 43 677). Comparison with the vitamin K-dependent plasma proteins reveals an extensive homology. The N-terminal part, containing 13 gamma-carboxyglutamic acid and one beta-hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid-102 is present. Histidine-57 and serine-195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.     

Crystal structures of A-DNA duplexes

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G · T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G · ^m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G · C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology. © 1997 John Wiley & Sons, Inc. Biopoly 44: 45–63, 1997     

Alternative conformations of DNA modified by N-2-acetylaminofluorene

Modification of DNA by the carcinogen N-acetoxy-N-2-acetylaminofluorene gives two adducts, a major one at the C-8 position of guanine and a minor one at the N-2 position with differing conformations. Binding at the C-8 position results in a large distortion of the DNA helix referred to as the “base displacement model” with the carcinogen inserted into the DNA helix and the guanosine displaced to the outside. The result is increased susceptibility to nuclease S, digestion due to the presence of large, single-stranded regions in the modified DNA. In contrast, the N-2 adduct results in much less distortion of the helix and is less susceptible to nuclease S₁ digestion. A third and predominant adduct is formed in vivo, the deacetylated C-8 guanine adduct. The conformation of this adduct has been investigated using the dimer dApdG as a model for DNA. The attachment of aminofluorene (AF) residues introduced smaller changes in the circular dichroism (CD) spectra of dApdG than binding of acetylaminofluorene (AAF) residues. Similarly, binding of AF residues caused lower upfield shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with neighboring bases than AAF and induce less distortion in conformation of the modified regions than AAF. An alternative conformation of AAF-modified deoxyguanosine has been suggested based on studies of poly(dG-dC)·poly(dG-dC). Modification of this copolymer with AAF to an extent of 28% showed a CD spectrum that had the characteristics of the left-handed Z conformation seen in unmodified poly-(dG-dC)·poly(dG-dC) at high ethanol or salt concentrations. Poly(dG)·poly(dC), which docs not undergo the B to Z transition at high ethanol concentrations, did not show this type of conformational change with high AAF modification. Differences in conformation were suggested by single-strand specific nuclease S₁ digestion and reactivity with anticytidine antibodies. Highly modified poly(dG-dC)·poly(dG-dC) was almost completely resistant to nuclease S₁ hydrolysis, while, modified DNA and poly(dG)·poly(dC) are highly susceptible to digestion. Two possible conformations for deoxyguanosine modified at the C-8 position by AAF are compared depending on whether its position is in alternating purine-pyrimidine sequences or random sequence DNA.     

Characteristics of Z-DNA helices formed by imperfect (purine-pyrimidine) sequences in plasmids

The capacities of three synthetic sequences to adopt left-handed helices were evaluated in recombinant plasmids. The sequences consisted of very short runs of (CG)n (n = 2-4) interspersed with runs of alternating A.T base pairs and/or with regions of non-alternating base pairs. The plasmids were studied by two-dimensional gel electrophoresis to determine the natures of the conformational transitions and their free energies of formation. These results coupled with analyses with chemical (diethyl pyrocarbonate, osmium tetroxide, and bromoacetaldehyde) and enzymatic (S1 nuclease, T7 gene 3 product, and MHhaI) probes indicated that the entire sequence was adopting a left-handed helix in each case. In one of these sequences, Z-DNA formation necessitated the retention of the anti conformation of one of the guanines in a region of non-alternation. In a sequence which contains out-of-phase regions of alternation, our results indicate the formation of a separate left-handed helix in the central (CG)2 region, thus forming two Z-Z junctions. In summary, we conclude that only very short regions of alternating CG are necessary to effect the B to Z transition and that this conformational change can be transmitted through non-alternating regions. A set of empirical rules governing the characteristics of the B to Z transition and the types of left-handed helices in supercoiled plasmids was derived from studies on a systematic series of 17 plasmids.     

First Principles Modeling of Dissociative Adsorption at Crystal Surfaces: Hydrogen on Pt(111)

Multiscale simulation has the potential of becoming the new modeling paradigm in chemical sciences. An important class of multiscale models involves the mapping of a finer scale model into an approximate surface that is used by a coarser scale model. As a specific example of this class we present the case of the adsorption dynamics of diatomic molecules on single crystal catalyst surfaces. The prototype system studied is the dissociative adsorption of H₂ on Pt(111). The finer scale model consists of density functional theory (DFT) periodic slab calculations that provide a small dataset for training an atomistic scale potential energy surface. The coarser scale model uses a semi-classical molecular dynamics (MD) algorithm to obtain the sticking coefficient as a function of the incident energy. Comparison to experimental data and published simulation work is presented. Finally, major challenges in multiscale modeling of chemical reactivity in coupled DFT/MD simulations are discussed, specifically the need for a systematic method of assessing the accuracy of the coarse graining process.