DNA fine structure and dynamics in crystals and in solution: the impact of BI/BII backbone conformations

Sugar-phosphate backbone conformations are an important structural element for a complete understanding of specific recognition in nucleic acid-protein interactions. They can be involved both in early stages of target discrimination and in structural adaptation upon binding. In the first part of this study, we have analyzed high-resolution structures of double-stranded B-DNA either isolated or bound to proteins, and explored the impact of both the standard BI and the unusual BII phosphate backbone conformations on neighboring sugar puckers and on selected helical parameters. Correlations are found to be similar for free and bound DNA, and in both categories, the possible facing backbone conformations (BI.BI, BI.BII, and BII.BII) define well-characterized substates in the B-DNA conformational space. Notably, BII.BII steps are characterized by specific, and sequence-independent, structural effects involving reduced standard deviations for almost all conformational parameters. In the second part of this work, we analyze four 10 ns molecular dynamics simulations in explicit solvent on the DNA targets of NF-kappaB and bovine papillomavirus E2 proteins, highlighting the multiplicity of backbone dynamical behavior. These results show sequence effects on the percentages of BI and BII conformers, the preferential state of facing backbones, the occurrence of coupled transitions. The backbone states can consequently be seen as a mechanism for transmitting information from the bases to the phosphate groups and thus for modulating the overall structural properties of the target DNA.     

Interaction specificity of violamycin BI and BII with DNA using the hyperchromic spectra method

Interaction specificity of the anthracycline antibiotics violamycin BI and violamycin BII in respect to A.T and G.C pairs was investigated. For comparison denaturation of complexes with A.T and G.C specific ligands distamycin A and actinomycin D are presented. Making use of the least squares hyperchromic spectra measured in the course of thermal denaturation were partitioned into the components corresponding to the melting of A.T and G.C base pairs and dissociation of ligand. The mutual dependence of AT and GC denaturation allows one to draw conclusions about specificity of interaction. In case of both violamycins only slight preference of interaction with AT-rich regions was detected. The dissociation of violamycin BII in the latest stage of thermal denaturation was found to be cooperative.     

Tricholongins BI and BII, 19-residue peptaibols from Trichoderma longibrachiatum. Solution structure from two-dimensional NMR spectroscopy

Two nonadecapeptides, tricholongins BI and BII, which display antifungal and antibacterial activities, have been isolated from in vitro cultures of the fungus Trichoderma longibrachiatum. The peptides were separated by reversed-phase HPLC; their amino acid compositions were determined by gas chromatography and their sequences by positive-ion fast-atom-bombardment mass spectrometry and high-field NMR. These linear peptides, containing mainly hydrophobic L-amino acids, 8-9 2-aminoisobutyric acid residues and exhibiting an acetylated N-terminal residue and an amino alcohol C-terminal leucinol belong to the peptaibol class. The methanol solution structure of tricholongins BI and BII has been investigated using both one- and two-dimensional NMR techniques. The total 1H-NMR and 13C-NMR assignments are given. By a combination of the 3JNH,C alpha H coupling constant values, temperature coefficients of the NH and CO groups, amide hydrogen/deuterium-exchange rate measurements and NOE data, a secondary structure for tricholongins in solution has been proposed. Both peptides adopt a similar alpha-helical conformation with a hinge around Pro13 resulting from two 3(10) bonds. The results suggest that the N-terminus contains mixed alpha/3(10) bonds. The membrane permeability modifications induced by tricholongins have been assayed by the use of liposomes composed of egg phosphatidylcholine with 20-30% cholesterol. The peptide-induced leakage of an entrapped fluorescent probe has been followed by fluorescence spectroscopy. In a concentration range of 0.13-0.31 microM, tricholongins induce the leakage of 50% of the entrapped material in 20 min.     

Solid-state conformations of aminosuccinyl peptides: crystal structure of tert-butyloxycarbonyl-L-leucyl-L-aminosuccinyl-L-phenylalaninami de

The protected tripeptide tert-butyloxycarbonyl-L-leucyl-L-aminosuccinyl-L-phenylalaninamide crystallizes in the orthorhombic space group P2(1)2(1)2(1), with a = 6.214(3), b = 12.832(3), c = 33.094(4) A, Z = 4. The structure was solved by direct methods using MULTAN 80 and refined to an R value of 0.055 for 1458 reflections. The bond lengths and angles are in good agreement with the standard values. The peptide backbone adopts a type II' beta-bend conformation with a weak intramolecular hydrogen bond between the CO group of the leucyl residue and the C-terminal NH2 group. In agreement with previous studies, this structure confirms the high propensity of aminosuccinyl peptides to adopt a type II' beta-bend conformation. The role of this conformation in relation to the deamidation process in proteins is also discussed.     

Sequence analysis by additive scales: DNA structure for sequences and repeats of all lengths

MOTIVATION: DNA structure plays an important role in a variety of biological processes. Different di- and tri-nucleotide scales have been proposed to capture various aspects of DNA structure including base stacking energy, propeller twist angle, protein deformability, bendability, and position preference. Yet, a general framework for the computational analysis and prediction of DNA structure is still lacking. Such a framework should in particular address the following issues: (1) construction of sequences with extremal properties; (2) quantitative evaluation of sequences with respect to a given genomic background; (3) automatic extraction of extremal sequences and profiles from genomic databases; (4) distribution and asymptotic behavior as the length N of the sequences increases; and (5) complete analysis of correlations between scales. RESULTS: We develop a general framework for sequence analysis based on additive scales, structural or other, that addresses all these issues. We show how to construct extremal sequences and calibrate scores for automatic genomic and database extraction. We show that distributions rapidly converge to normality as Nincreases. Pairwise correlations between scales depend both on background distribution and sequence length and rapidly converge to an analytically predictable asymptotic value. For di- and tri-nucleotide scales, normal behavior and asymptotic correlation values are attained over a characteristic window length of about 10-15 bp. With a uniform background distribution, pairwise correlations between empirically-derived scales remain relatively small and roughly constant at all lengths, except for propeller twist and protein deformability which are positively correlated. There is a positive (resp. negative) correlation between dinucleotide base stacking (resp. propeller twist and protein deformability) and AT-content that increases in magnitude with length. The framework is applied to the analysis of various DNA tandem repeats. We derive exact expressions for counting the number of repeat unit classes at all lengths. Tandem repeats are likely to result from a variety of different mechanisms, a fraction of which is likely to depend on profiles characterized by extreme structural features.     

Geometry of proline and hydroxyproline I: An analysis of X-ray crystal structure data

We have carried out an analysis of crystal structure data on prolyl and hydroxyprolyl moieties in small molecules. The flexibility of the pyrrolidine ring due to the pyramidal character of nitrogen has been defined in terms of two projection angles delta 1 and delta 2. The distribution of these parameters in the crystal structures is found to be consistent with results of the energy calculations carried out on prolyl moieties in our laboratory.     

Sequence dependent DNA conformations: Raman spectroscopic studies and a model of action of restriction enzymes

Raman spectra have been examined to clarify the polymorphic forms of DNA, A, B, and Z forms. From an analysis we found that the guanine ring breathing vibration is sensitive to its local conformation. Examination of nine crystals of guanosine residues in which the local conformations are well established revealed that a guanosine residue with a C3′endo-anti gives a strong line at 666±2cm⁻¹, O4′endo-anti at 682 cm⁻¹, Cl′exo-anti at 673 cm⁻¹, C2′endo-anti at 677 cm⁻¹ and syn-forms around 625 cm⁻¹. Using this characteristic line, we were able to obtain the local conformations of guanosine moieties in poly(dG-dC).Such a sequence derived variation is suggested to be recognized by sequence specific proteins such as restriction enzymes. We found a correlation between sequence dependent DNA conformation and a mode of action of restriction enzymes. The cutting mode of restriction enzymes is classified into three groups. The classification of whether the products have blunt ends, two-base-long cohesive ends, or four-base-long cohesive ends depends primarily on the substrate, not on the enzyme. It is suggested that sequence dependent DNA conformation causes such a classification by the use of the Calladine-Dickerson analysis. In the recognition of restriction enzymes, the methyl group in a certain sequence is considered to play an important role by changing the local conformation of DNA.     

家鹅线粒体tRNAthr与tRNApro序列与结构分析

本研究采用PCR和DNA测序技术测定了6个中国家鹅品种和2个欧洲鹅品种25个个体线粒体DNA tRNApro（69bp）和tRNAthr（68bp）基因的完整序列,通过对家鹅线粒体基因组的研究,首次报道了家鹅线粒体tRNApro和tRNAthr基因的结构,对鸿雁家鹅、灰雁家鹅、白额雁（Anser albifrons,序列号为AF363031）种间的tRNApro和tRNAthr基因的二级结构及序列的变异特征进行了分析,并通过家鸡（Gallus gallus domesticus,序列号为NC001323）与鸿雁家鹅间tRNApro和tRNAthr基因二级结构的比较,初步进行了鸡形目与雁形目两个目间tRNApro和tRNAthr基因二级结构及序列变异的分析。结果表明：家鹅tRNApro和tRNAthr基因均可折叠成标准的三叶草形二级结构; 2个tRNA基因三叶草结构的氨基酸臂、反密码子环在鸿雁、灰雁和白额雁种间以及鸡形目与雁形目两个目间没有发生变异,具有高度的保守性。本研究的结果将为进一步探讨家鹅线粒体DNA tRNApro和tRNAthr基因序列与结构、功能的关系奠定基础。所测定的基因序列已登录国际GenBank数据库,序列号为AY427800～AY427805和AY427812～AY427814。     

Sequence context dependence of tandem guanine:adenine mismatch conformations in RNA: a continuum solvent analysis

Guanine:adenine (G:A) mismatches and in particular tandem G:A (tG:A) mismatches are frequently observed in biological RNA molecules and can serve as sites for tertiary interaction, metal binding and protein recognition. Depending on the surrounding sequence tG:A mismatches can adopt different basepairing topologies. In the sequence context (5'-) GGAC (tandem G:A in bold) a face-to-face (imino or Watson-Crick-like) pairing is preferred whereas in the CGAG context, G and A adopt a sheared arrangement. Systematic conformational searches with a generalized Born continuum model and molecular dynamics simulations including explicit water molecules and ions have been used to generate face-to-face and sheared tG:A mismatches in both CGAG and GGAC sequence contexts. Conformations from both approaches were evaluated using the same force field and a Poisson-Boltzmann continuum solvent model. Although the substate analysis predicted the sheared arrangement to be energetically preferred in both sequence contexts, a significantly greater preference of the sheared form was found for the CGAG context. In agreement with the experimental observation, the analysis of molecular dynamics trajectories indicated a preference of the sheared form in the case of the CGAG-context and a favorization of the face-to-face form in the case of the GGAC context. The computational studies allowed to identify energetic contributions that stabilize or destabilize the face-to-face and sheared tandem mismatch topologies. The calculated nonpolar solvation and Lennard-Jones packing interaction were found to stabilize the sheared topology independent of the sequence context. Electrostatic contributions are predicted to make the most significant contribution to the sequence context dependence on the structural preference of tG:A mismatches.     

The Alzheimer's peptides Abeta40 and 42 adopt distinct conformations in water: a combined MD / NMR study

The role of peptides Abeta40 and Abeta42 in the early pathogenesis of Alzheimer's disease (AD) is frequently emphasized in the literature. It is known that Abeta42 is more prone to aggregation than Abeta40, even though they differ in only two (IA) amino acid residues at the C-terminal end. A direct comparison of the ensembles of conformations adopted by the monomers in solution has been limited by the inherent flexibility of the unfolded peptides. Here, we characterize the conformations of Abeta40 and Abeta42 in water by using a combination of molecular dynamics (MD) and measured scalar (3)J(HNHalpha) data from NMR experiments. We perform replica exchange MD (REMD) simulations and find that classical forcefields reproduce the NMR data quantitatively when the sampling is extended to the microseconds time-scale. Using the quantitative agreement of the NMR data as a validation of the model, we proceed to compare the conformational ensembles of the Abeta40 and Abeta42 peptide monomers. Our analysis confirms the existence of structured regions within the otherwise flexible Abeta peptides. We find that the C terminus of Abeta42 is more structured than that of Abeta40. The formation of a beta-hairpin in the sequence (31)IIGLMVGGVVIA involving short strands at residues 31-34 and 38-41 (in bold) reduces the C-terminal flexibility of the Abeta42 peptide and may be responsible for the higher propensity of this peptide to form amyloids.     

Sequence of lamprey vitellogenin. Implications for the lipovitellin crystal structure.

The amino acid sequence of lamprey vitellogenin has been predicted from the nucleotide sequence of cloned cDNA. The sites of proteolytic cleavage that produce the lipovitellin complex from the vitellogenin have been located by comparing the N-terminal sequences of two lamprey lipovitellin polypeptides with the predicted sequence. These results also confirm that the vitellogenin sequence derived here corresponds to the lipovitellin complex for which the crystal structure has been solved previously. Predictions of secondary structure indicate that the region most likely to correspond to the large alpha-helical domain of the crystallographic model consists of vitellogenin residues 300 to 600. Similar to the lipovitellins of Xenopus laevis, lamprey lipovitellin appears to lack approximately 200 C-terminal residues that are present in vitellogenin. However, the lamprey lipovitellin differs from those of Xenopus and chicken in two respects. First, most of the serine-rich domain that is present as the phosvitin polypeptide in the lipovitellins of the higher vertebrates appears to be lost in the maturation of lamprey vitellogenin to lipovitellin. Second, the domains that constitute the large lipovitellin-1 polypeptide in Xenopus and chicken are present in two polypeptides in lamprey, owing to an additional proteolytic processing event.     

A refined prediction method for gel retardation of DNA oligonucleotides from dinucleotide step parameters: reconciliation of DNA bending models with crystal structure data

The development and assessment of a prediction method for gel retardation and sequence dependent curvature of DNA based on dinulcleotide step parameters are described. The method is formulated using the Babcock-Olson equations for base pair step geometry (1) and employs Monte Carlo simulated annealing for parameter optimization against experimental data. The refined base pair step parameters define a stuctural construct which, when the width of observed parameter distributions is taken into account, is consistent with the results of DNA oligonucleotide crystal structures. The predictive power of the method is demonstrated and tested via comparisons with DNA bending data on sets of sequences not included in the training set, including A-tracts with and without periodic helix phasing, phased A4T4 and T4A4 motifs, a sequence with a phased GGGCCC motif, some "unconventional" helix phasing sequences, and three short fragments of kinetoplast DNA from Crithidia fasiculata that exhibit significantly different behavior on non-denaturing polyacrylamide gels. The nature of the structural construct produced by the methodology is discussed with respect to static and dynamic models of structure and representations of bending and bendability. An independent theoretical account of sequence dependent chemical footprinting results is provided. Detailed analysis of sequences with A-tract induced axis bending forms the basis for a critical discussion of the applicability of wedge models,junction models and non A-tract, general sequence models for understanding the origin of DNA curvature at the molecular level.     

MultiSeq: unifying sequence and structure data for evolutionary analysis

Background  

Since the publication of the first draft of the human genome in 2000, bioinformatic data have been accumulating at an overwhelming pace. Currently, more than 3 million sequences and 35 thousand structures of proteins and nucleic acids are available in public databases. Finding correlations in and between these data to answer critical research questions is extremely challenging. This problem needs to be approached from several directions: information science to organize and search the data; information visualization to assist in recognizing correlations; mathematics to formulate statistical inferences; and biology to analyze chemical and physical properties in terms of sequence and structure changes.     

CH/pi interactions in the crystal structure of TATA-box binding protein/DNA complexes

Crystal structures of TATA box-binding proteins (TBP) of various sources bound to their promoter DNA (TATA box) were analyzed with use of our program CHPI. A number of short CH/Csp2 contacts have been unveiled in these complexes at the boundary of TBP and the TATA box minor groove. The result was discussed in the context of the CH/pi interaction. Thus, the nature of nonpolar forces, reported in the past at the interface of the two components, has been attributed to the CH/pi interaction. Furthermore, many CH/pi contacts have been disclosed within the same strand of the promoter DNA. The structure of the TATA element, partially unwound and severely bent on complexation, seems to be stabilized by CH/pi interactions; H2' of the deoxyribose moiety and the methyl group in the thymine nucleotide play the primary role.     

BII nucleotides in the B and C forms of natural-sequence polymeric DNA: A new model for the C form of DNA

A combination of solid-state (31)P and (13)C NMR, X-ray diffraction, and model building is used to show that the B and C forms of fibrous macromolecular DNA consist of two distinct nucleotide conformations, which correspond closely to the BI and BII nucleotide conformations known from oligonucleotide crystals. The proportion of the BII conformation is higher in the C form than in the B form. We show structural models for a 10(1) double helix involving BI nucleotides and a 9(1) double helix involving BII nucleotides. The 10(1) BI model is similar to a previous model of B-form DNA, while the 9(1) BII model is novel. The BII model has a very deep and narrow minor groove, a shallow and wide major groove, and highly inclined bases. This work shows that the B to C transition in fibers corresponds to BI to BII conformational changes of the individual nucleotides.     

Design and analysis of multiple choice feeding preference data

Traditional analyses of feeding experiments that test consumer preference for an array of foods suffer from several defects. We have modified the experimental design to incorporate into a multivariate analysis the variance due to autogenic change in control replicates. Our design allows the multiple foods to be physically paired with their control counterparts. This physical proximity of the multiple food choices in control/experimental pairs ensures that the variance attributable to external environmental factors jointly affects all combinations within each replicate. Our variance term, therefore, is not a contrived estimate as is the case for the random pairing strategy proposed by previous studies. The statistical analysis then proceeds using standard multivariate statistical tests. We conducted a multiple choice feeding experiment using our experimental design and utilized a Monte Carlo analysis to compare our results with those obtained from an experimental design that employed the random pairing strategy. Our experimental design allowed detection of moderate differences among feeding means when the random design did not.     

Charge structure and counterion distribution in hexagonal DNA liquid crystal

A hexagonal liquid crystal of DNA fragments (double-stranded, 150 basepairs) with tetramethylammonium (TMA) counterions was investigated with small angle neutron scattering (SANS). We obtained the structure factors pertaining to the DNA and counterion density correlations with contrast matching in the water. Molecular dynamics (MD) computer simulation of a hexagonal assembly of nine DNA molecules showed that the inter-DNA distance fluctuates with a correlation time around 2 ns and a standard deviation of 8.5% of the interaxial spacing. The MD simulation also showed a minimal effect of the fluctuations in inter-DNA distance on the radial counterion density profile and significant penetration of the grooves by TMA. The radial density profile of the counterions was also obtained from a Monte Carlo (MC) computer simulation of a hexagonal array of charged rods with fixed interaxial spacing. Strong ordering of the counterions between the DNA molecules and the absence of charge fluctuations at longer wavelengths was shown by the SANS number and charge structure factors. The DNA-counterion and counterion structure factors are interpreted with the correlation functions derived from the Poisson-Boltzmann equation, MD, and MC simulation. Best agreement is observed between the experimental structure factors and the prediction based on the Poisson-Boltzmann equation and/or MC simulation. The SANS results show that TMA is too large to penetrate the grooves to a significant extent, in contrast to what is shown by MD simulation.     

Sequence and structure of epoxide hydrolases: a systematic analysis

Epoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. More than 100 epoxide hydrolases (EH) have been identified or predicted, and 3 structures are available. Although they catalyze the same chemical reaction, sequence similarity is low. To identify conserved regions, all EHs were aligned. Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs. Bacterial EHs show a high sequence diversity. Based on structure comparison of three known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed. Although core and cap domain are highly conserved, the structural differences between the EHs are restricted to only two loops: the NC-loop connecting the core and the cap and the cap-loop, which is inserted into the cap domain. EHs were assigned to either of three clusters based on loop length. By using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled. Representative models are available from the Lipase Engineering Database, http://www.led.uni-stuttgart.de.     

Cobalt complexes of terpyridine ligand: crystal structure and photocleavage of DNA

Two new cobalt complexes, [Co(pytpy)(2)](ClO(4))(2), 1, and [Co(pytpy)(2)](ClO(4))(3), 2 where pytpy=pyridine terpyridine, have been synthesized and characterized. Single-crystal X-ray structure of both the complexes has been resolved. The structure shows the complexes to be a monomeric cobalt(II) and cobalt(III) species with two pytpy ligands coordinated to the metal ion to give a six coordinate complex. Both cobalt(II) and cobalt(III) complexes crystallize in meridional configuration. The interaction of these complexes with calf thymus DNA has been explored by using absorption, emission spectral, electrochemical studies and viscosity measurements. From the experimental results the DNA binding constants of 1 and 2 are found to be (1.97+/-0.15)x10(4)M(-1) and (2.7+/-0.20)x10(4)M(-1) respectively. The ratio of DNA binding constants of 1 and 2 have been estimated to be 0.82 from electrochemical studies, which is in close agreement with the value of 0.73 obtained from spectral studies. The observed changes in viscosity of DNA in the presence of increasing amount of complexes 1 and 2 suggest intercalating binding of these complexes to DNA. Results of DNA cleaving experiments reveal that complex 2 efficiently cleaves DNA under photolytic conditions while complex 1 does not cleave DNA under similar conditions.