Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites

A 4'-oxidized abasic site (X) has been synthesized in a defined duplex DNA sequence, 5'-d(CCAAAGXACCGGG)-3'/3'-d(GGTTTCATGGCCC)-5' (1). Its structure has been determined by two-dimensional NMR methods, molecular modeling, and molecular dynamics simulations. 1 is globally B-form with the base (A) opposite X intrahelical and well-stacked. Only the alpha anomer of X is observed, and the abasic site deoxyribose is largely intrahelical. These results are compared with a normal abasic site (Y) in the same sequence context (2). Y is composed of a 60:40 mixture of alpha and beta anomers (2alpha and 2beta). In both 2alpha and 2beta, the base (A) opposite Y is intrahelical and well-stacked and the abasic site deoxyribose is predominantly extrahelical, consistent with the reported structures of the normal abasic site in a similar sequence context [Hoehn, S. T., Turner, C. J., and Stubbe, J. (2001) Nucleic Acids Res. 29, 3413-3423]. Molecular dynamics simulations reveal that the normal abasic site appears to be conformationally more flexible than the 4'-oxidized abasic site. The importance of the structure and flexibility of the abasic site in the recognition by the DNA repair enzyme Ape1 is discussed.     

Nuclear magnetic resonance studies of the snake toxin echistatin. 1H resonance assignments and secondary structure.

The 1H-NMR spectrum of the snake toxin echistatin has been assigned using homonuclear two-dimensional methods. Consideration of the NOE patterns, coupling constants and putative hydrogen bonds enabled two regular features of secondary structure to be deduced: a beta-sheet/turn between residues 8 and 13 and a small anti-parallel beta-sheet and bulge linking residues 16-20 with residues 30-33. The recognition region of the protein containing the residues RGD lies in a loop joining the two strands of the beta-sheet. The beta-bulge and the loop containing the RGD sequence undergo pH-dependent conformational interconversion, modulated by the side chain of Asp29.     

Nuclear magnetic resonance studies of amphiphile hydration.

The nuclear magnetic resonance signal of water which remains unfrozen at ?25 °C in the presence of phosphatidylcholine has been used to determine the hydration of this amphiphile. The effects of cholesterol and sodium dodecylsulfate on both the area and linewidth of this signal indicate that these molecules cause significant changes in the structure of phosphatidylcholine vesicles in solution. Studies on other amphiphiles indicate that, whereas phosphatidylethanolamine has a hydration similar to phosphatidylcholine, species with just one hydrocarbon chain such as sodium dodecylsulfate and dodecyltrimethylammonium bromide have little, if any, hydration when assayed via the nuclear magnetic resonance experiment.     

Multinuclear magnetic resonance studies of collagen molecular structure and dynamics

We have measured the percentages of cis and trans Gly-Pro and X-Hyp peptide bonds in thermally unfolded type I collagen. ¹³C-nmr solution spectra show that 16% of the Gly-Pro and 8% of the X-Hyp bonds are cis in unfolded chick calvaria collagen. These results support the hypothesis that cis–trans isomerization is that rate-limiting step in the propagation of the collagen triple helix. We have used multinuclear solid-state nmr to study the molecular dynamics of the collagen backbone in tendon, demineralized bone, and intact bone as a function of temperature, hydration, and pH. These studies show that collagen backbone motions are characterized by a broad distribution of correlation times, τ, covering the range from 10^?4 to 10^?9 s. In the case of nonmineralized collagen, the root-mean-square fluctuations in azimuthal angle, γ_rms, range from ca. 10° when τ ～ 10^?9 s to ca. 30° when τ < 10^?4 s; in the case of bone collagen, γ_rms values are about half as large as those found in nonmineralized collagen. Backbone motions are negligible at temperatures below ?25°C. This is also the case at 22°C when demineralized bone collagen is lyophilized. In contrast, flexibility of hydrated demineralized bone collagen greatly increases as pH is lowered from 7 to 2. The more limited flexibility observed at neutral pH is a consequence of the intermolecular interactions that contribute to fibril organization and strength. However, the fibrils retain significant flexibility at physiological pH, enabling them to distribute stress and dissipate mechanical energy.     

Nuclear magnetic resonance studies of residual structure in thermally unfolded ribonuclease A.

A proton nuclear magnetic resonance study of the four histidine residues of thermally unfolded ribonuclease A has provided evidence that two of the residues are in regions of residual structure, whereas the other two are freely exposed to solvent. Histidine-48 and, tentatively, histidine-105 occupy an environment at 69 degrees characterized by residual structure and display a pK value of 5.75 and a spin-lattice relaxation time of about 0.8 sec at pH 5.5. Histidine-12 and, tentatively, histidine-119 are in an environment at 69 degrees which is freely accessible to solvent and show a pK value of 5.96 and a spin-lattice relaxation time of about 1.1 sec at pH 5.5.     

Nuclear magnetic resonance studies on the structure of the tetrapeptide tuftsin, L-threonyl-L-lysyl-L-prolyl-L-arginine, and its pentapeptide analogue L-threonyl-L-lysyl-L-prolyl-L-prolyl-L-arginine.

Nuclear magnetic resonance spectroscopy has been used to investigate the solution conformation of tuftsin, threonyllysylprolylarginine, as well as a pentapeptide inhibitor of tuftsin, threonyllysylprolylprolylarginine. Both proton and carbon-13 studies were performed. In water, neither peptide gives evidence of a preferred conformation. In dimethyl-d6 sulfoxide, tuftsin appears to prefer a particular conformation, but the inhibitor does not. The conformation of tuftsin is one in which the amide NH proton of arginine is solvent shielded. The conformation does not, however, appear to be such that a normal 4 leads to 1 beta turn exists.     

Nuclear magnetic resonance studies of the denaturation of ubiquitin.

The effects of pH, temperature and guanidine hydrochloride concentration on the structure of ubiquitin, a polypeptide which can activate adenylate cyclase and can mimic thymopoietin induced differentiation of prothymocytes, were monitored using nuclear magnetic resonance spectroscopy. This relatively small polypeptide (molecular weight of 8541) exhibits a remarkable stability towards pH and temperature changes. At 7 M guanidine hydrochloride concentration, the structure of ubiquitin is essentially a random coil.     

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.     

Nuclear magnetic resonance and molecular genetic studies of the membrane-bound D-lactate dehydrogenase of Escherichia coli

In this study we demonstrate the potential of combining fluorine-19 nuclear magnetic resonance (NMR) spectroscopy with molecular genetics. We are using the membrane-bound enzyme D-lactate dehydrogenase of Escherichia coli as a model system to characterize interactions between proteins and lipids. We have labeled D-lactate dehydrogenase with 4-, 5-, and 6-fluorotryptophans and obtained high-resolution fluorine-19 NMR spectra showing five resonances, in agreement with the five tryptophan residues expected from the DNA sequence. The five 19F resonances in the spectra have been assigned to the specific tryptophan residues in the primary sequence of D-lactate dehydrogenase by site-directed oligonucleotide mutagenesis of the cloned gene. We observe large differences in the relative fluorine-19 chemical shifts of each tryptophan residue when labeled by different isomers of fluorotryptophan. We have determined by NMR methods that two tryptophans are exposed to the solvent and that none of the tryptophan residues are within 10 A of the lipid phase. On the basis of 19F NMR spectroscopy of the labeled tryptophan residues, the conformation of D-lactate dehydrogenase is similar in aqueous solution and in the presence of a variety of lipids and detergents. This result indicates that the presence of lipids or detergents is not required to maintain the tertiary structure of this membrane-bound enzyme. In contrast, Triton X-100 induces a change to an abnormal conformation of the enzyme as judged from both NMR spectroscopy and the effect of temperature on the maximal velocity of the enzyme in the presence of this detergent.     

Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1:1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C.G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.     

Conformational properties of DNA dodecamers containing four tandem repeats of the CNG triplets.

We studied DNA dodecamers (CAG)4, (CCG)4, (CGG)4 and (CTG)4by CD spectroscopy and polyacrylamide gel electrophoresis. Each dodecamer adopted several ordered conformers which denatured in a cooperative way. Stability of the conformers depended on the dodecamer concentration, ionic strength, temperature and pH. The dodecamers, having a pyrimidine base in the triplet center, generated foldbacks at low ionic strength whose stem conformations were governed by the GC pairs. At high salt, (CCG)4 isomerized into a peculiar association of two strands. The association was also promoted by high oligonucleotide concentrations. No similar behavior was exhibited by (CTG)4. At low salt, (CGG)4 coexisted in two bimolecular conformers whose populations were strongly dependent on the ionic strength. In addition, (CGG)4 associated into a tetraplex at acidic pH. A tetraplex was even observed at neutral pH if the (CGG)4 concentration was sufficiently high. (CAG)4 was very stable in a monomolecular conformer similar to the known extremely stable foldback of the (GCGAAGC) heptamer. Nevertheless, even this very stable conformer disappeared if (CTG)4 was added to the solution of (CAG)4. Association of the complementary strands was also strongly preferred to the particular strand conformations by the other couple, (CCG)4 and (CGG)4.     

Hairpin structures in DNA containing arabinofuranosylcytosine. A combination of nuclear magnetic resonance and molecular dynamics

Nuclear magnetic resonance (NMR) and model-building studies were carried out on the hairpin form of the octamer d(CGaCTAGCG) (aC = arabinofuranosylcytosine), referred to as the TA compound. The nonexchangeable protons of the TA compound were assigned by means of nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY). From a detailed analysis of the coupling data and of the NOESY spectra the following conclusions are reached: (i) The hairpin consists of a stem of three Watson-Crick type base pairs, and the two remaining residues, T(4) and dA(5), participate in a loop. (ii) All sugar rings show conformational flexibility although a strong preference for the S-type (C2'-endo) conformer is observed. (iii) The thymine does not stack upon the 3' side of the stem as expected, but swings into the minor groove. (This folding principle of the loop involves an unusual alpha t conformer in residue T(4).) (iv) At the 5'-3' loop-stem junction a stacking discontinuity occurs as a consequence of a sharp turn in that part of the backbone, caused by the unusual beta + and gamma t torsion angles in residue dG(6). (v) The A base slides over the 5' side of the stem to stack upon the aC(3) residue at the 3' side of the stem in an antiparallel fashion. On the basis of J couplings and a set of approximate proton-proton distances from NOE cross peaks, a model for the hairpin was constructed. This model was then refined by using an iterative relaxation matrix approach (IRMA) in combination with restrained molecular dynamics calculations. The resulting final model satisfactorily explains all the distance constraints.     

The structure of two alanine containing ferrichromes: Sequence determination by proton magnetic resonance

Summary Metal coordination confers an extraordinary structural stability to the ferrichromes which, independent of their variable amino acid composition, results in a basically unperturbed conformation for all the homologous peptides in the series. The proton magnetic resonance (pmr) characteristics for Al³⁺ analogues (alumichromes) reflect this conformational isomorphism in usual solvents so that single site substitutions are clearly recognized in the pmr spectra. Thus, the substitution of glycine byl-alanine orl-serine introduce new resonances characteristic of the sidechains and alter the pattern of the amide NH pmr region in that doublets substitute for glycyl triplets at the same site. Since for glycine- andl-serine-containing alumichromes the resonances have already been identified, it is possible to unequivocally establish the primary structure of the twol-alanyl homologues ferrichrome C ( ) and sake colorant A ( ) on the basis of the comparative pmr spectra of their Al³⁺ analogues, namely, alumichrome C and alumisake. The resonance assignment, and hence the site occupancy, is substantiated by the temperature coefficients of the NH chemical shifts, rates of¹H-²H exchange and homonuclear proton spin decoupling experiments centered on the NH spectral region. Occupancy of site 1 by a glycine residue is observed for all known ferrichromes, which serves to conserve a hairpin turn. This method of obtaining sequence information should prove of general use for other systems of homologous polypeptides, provided their conformations are not affected by the residue substitutions.     

Nuclear magnetic resonance studies of histone IV solution conformation.

The 220-MHz high-resolution proton magnetic resonance (PMR) spectrum of histone IV has been examined as a function of histone concentration, salt concentration, and pD. The hydrophobic C-terminal portion of the histone IV monomer appears to be largely PMR "invisible" indicating that this region of the polypeptide contains rigid secondary structure. Further loss of PMR resonance areas with increased histone IV concentration in neat D2O has been attributed to self-aggregation involving a monomer-dimer equilibrium. An equilibrium between the monomer and large aggregates, on the other hand, appears to dominate at NaCl concentrations above 0.01 M. pD studies reveal an abrupt increase in histone IV aggregation at pD smaller than 0.8 and precipitation of histone IV at pD values in the neighborhood of its isoelectric point, pD similar to 11.     

X-ray analyses of two DNA dodecamers containing N4-methoxy-cytosine paired with adenine or guanine.

To investigate mismatch of base-pairings in relation to mutagenesis by oxyamines, crystal structures of two DNA dodecamers with the sequence d(CGCZAA TTmo4CGCG) (Z = A or G), containing N4-methoxy-cytosine (mo4C), have been determined by X-ray analysis. These dodecamers essentially form right-handed B-form duplexes, respectively. In the dodecamer with Z = A, the two mo4C residues are adapted in imino form with the anti methoxyl group to form pairs with A on the opposite strand in a manner of Watson-Crick fashion. While in the dodecamer with Z = G, one mo4C in amino form with the anti methoxyl group forms a normal Watson-Crick pair with G, but the other one in imino form with syn methoxyl conformation wobbles with G. Based on these results, possible mutation mechanism has been proposed.