Discrimination Between A-type and B-type Conformations of Double Helical Nucleic Acid Fragments in Solution by Means of Two-dimensional Nuclear Overhauser Experiments

Abstract

The solution structure of two double helical nucleic acid fragments, viz. r(CGCGCG) and d(CGCGCG), was probed by means of two-dimensional nuclear Overhauser effect spectroscopy. The two compounds were selected as models for the A-type and B-type double helical conformations, respectively, and it is shown that for each of the two model compounds the intensities of the NOE cross peaks between base- and H2′ (deoxy)ribose proteins are qualitatively in correspondence with the relative NOE intensities expected on basis of the supposed duplex conformations. Thus our results indicate that NOE-data can be used to differentiate between A- and B-type double helical conformations in solution.

Coupling constant data show that, except for G(6), all ribose rings in r(CGCGCG) adopt pure N (C3′-endo) conformations thereby manifesting that this molecule takes up a regular A-type double helical conformation in solution. In contrast, the deoxyribose rings in d(CGCGCG) retain conformational freedom in the duplex state, albeit that the N/S- equilibrium is biased towards the S (C2′-endo) sugar conformation. This finding indicates that in solution the B-DNA backbone is highly dynamic.     

Discrimination between A-type and B-type conformations of double helical nucleic acid fragments in solution by means of two-dimensional nuclear Overhauser experiments

The solution structure of two double helical nucleic acid fragments, viz, r(CGCGCG) and d(CGCGCG), was probed by means of two-dimensional nuclear Overhauser effect spectroscopy. The two compounds were selected as models for the A-type and B-type double helical conformations, respectively, and it is shown that for each of the two model compounds the intensities of the NOE cross peaks between base- and H2' (deoxy)ribose proteins are qualitatively in correspondence with the relative NOE intensities expected on basis of the supposed duplex conformations. Thus our results indicate that NOE-data can be used to differentiate between A-and B-type double helical conformations in solution. Coupling constant data show that, except for G(6), all ribose rings in r(CGCGCG) adopt pure N (C3'-endo) conformations thereby manifesting that this molecule takes up a regular A-type double helical conformation in solution. In contrast, the deoxyribose rings in d(CGCGCG) retain conformational freedom in the duplex state, albeit that the N/S-equilibrium is biased towards the S (C2'-endo) sugar conformation. This finding indicates that in solution the B-DNA backbone is highly dynamic.     

Solution structure of RNA duplexes containing alternating CG base pairs: NMR study of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 under low salt conditions.

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.     

Salt induced conformational transition between A and Z forms of r(CGCGCG) as revealed by a Raman spectroscopic study

Solution conformation in different conditions of r(CGCGCG) has been studied by a Raman spectroscopic method. In NaCl solution, r (CGCGCG) takes only an A-form duplex in which guanosine and cytidine have C3'endo-anti conformation even at 5M salt concentration. In much higher ionic strength condition (5M NaCl plus 1M MgCl2 or 6M NaClO4), it undergoes a transition to a left-handed Z-form. The Raman spectrum of the Z-form RNA was found to be very similar to that of Z-form DNA, suggesting that Z-RNA involves a C3'endo-syn guanosine and an in between form of C2'endo-Cl'exo-anti cytidine.     

Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.     

Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).     

NMR studies of a bacterial cell culture medium (LB Broth): cyclic nucleosides in yeast extracts

The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H,31P-NMR spectroscopy, FPLC and gel electrophoresis. An unexpected finding was the high level of 2'3'-cyclic nucleotides, detected by characteristic 31P-NMR resonances in the region 20-21 ppm, originating from the yeast component. 31P-NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA.     

Influence of staphylococcal delta-toxin on the phosphatidylcholine headgroup as observed using 2H-NMR.

The interaction of the 8-toxin peptide isolated from Staphylococcus aureus with the headgroup region of lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy. At relatively low peptide/lipid ratios (P/L < 0.10), all 2H- and 31P-NMR spectral lineshapes at 25 degrees C were indicative of a single population of liquid-crystalline lipids in a bilayer arrangement. At these P/L ratios, delta-toxin had only marginal effects on the size of the quadrupole splitting measured from POPC labelled at either the alpha-methylene (POPC-alpha-d2) or the beta-methylene segment (POPC-beta-d2) of the choline headgroup and, similarly small effects on the magnitude of the chemical shift anisotropy (CSA) of the 31P-NMR spectrum. With increasing amounts of delta-toxin (0.10 < P/L < 0.15) the size of the 2H quadrupole splitting from POPC-alpha-d2, as well as the magnitude of the 31P-CSA, decreased progressively and rapidly. The quadrupole splitting from POPC-beta-d2, however, remained relatively unaffected. At yet higher levels of delta-toxin (P/L > 0.15), all 2H- and 31P-NMR spectra indicated the presence of multiple lipid populations experiencing varying degrees of increased conformational disordering. The spectral lineshapes of these apparently nonbilayer spectral components reverted to bilayer-type lineshapes upon lowering the measuring temperature to 5 degrees C. At the utmost highest level of delta-toxin measured here (P/L = 0.20), all 2H- and 31P-NMR spectra consisted of a single, broad, apparently nonbilayer-type component, indicative of hindered but virtual isotropic motional averaging of the POPC headgroups. In this case no reversion to bilayer-type spectra could be obtained by decreasing the temperature. We could obtain no evidence that the conformation of the choline headgroup of POPC was responding to any specific influence of delta-toxin on bilayer surface electrostatics.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase: roles of His 54, Asp130, Glu155, Lys185, and Aspl89

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 A resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670-22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5'-S(D) bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3'-oxidation and the C4'-proton abstraction are a concerted event and the C5'-S(D) bond cleavage is an independent event. The C4'-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3'-keto-4', 5'-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3'-oxidation, D130 abstracts the C4'-proton, D189, and E155 act as a communicator between the concerted C3'-oxidation and C4'-proton abstraction, and H54 plays as a general acid to cleave the C5'-S(D) bond of AdoHcy.     

Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies

In biological membranes, the anionic characteristics of the polar headgroup of phosphatidic acids are responsible for structural changes induced by Ca2+ in many cellular processes. The very simple headgroup structure of dipalmitoylphosphatidic acid (DPPA) offers particular advantages as a model to study the interactions between Ca2+ and natural phosphatidic acids such as cardiolipin and phosphatidylserine. The effects of calcium ions on DPPA membranes have been studied as a function of temperature by potentiometry and by Raman, ESR and 31P-NMR spectroscopies. The protons in monosodic DPPA liposomes have been considered as a probe to detect pH variations resulting from introduction of Ca2+ inside the membrane. This method has also allowed us to determine the stoichiometry of this reaction: 2 DPPA(H) + Ca2+----Ca(DPPA)2 + 2H+. 31P-NMR spectroscopy has been used to detect reorganization-condensation phenomena in multilamellar vesicles of DPPA under the influence of calcium and temperature. Furthermore, the temperature profiles obtained from Raman spectra for Ca(DPPA)2 membranes provide conclusive evidence that Ca2+ induces major reorganization of the phosphatidic acid component into a highly ordered phase. Quantitative estimates of the degree of motional restriction of spin-labeled soaps embedded inside membranes composed of DPPA with or without Ca2+ have been made using ESR technique. These results are discussed and compared to those found previously for a natural phosphatidic acids such as phosphatidylserine.     

Interaction of Et2SnCl2 with 5'-IMP and 5'-GMP

The interactions of Et2SnCl2 with 5'-IMP and 5'-GMP have been studied in aqueous solutions by 1H- and 31P-NMR spectroscopy as a function of pH. At low pH values (< 4.0) Sn(IV) interacts with the pyrophosphate oxygens of these nucleotides. At intermediate pH values (4-9.5) no interaction of the metal with the nucleotides take place, while at pH > 9.5 the sugar O'2 and O'3 atoms are the preferred coordination sites. In addition, the solid adducts obtained from aqueous solutions at pH = 3-4 of the above interactions correspond to formulae; (Et2Sn)2(5'-IMP)2(H2O) and (Et2Sn)3(5'-GMP)2(OH)2(H2O)2 as their elemental analysis show. IR spectra and solid state 13C, 31P-NMR spectra 119Sn M?ssbauer and solution 119Sn-NMR spectra once more confirm the pyrophosphate involvement in bonding with Sn(IV) in oligomeric or polymeric structures and trigonal bipyramidal or octahedral geometries.     

Solid-phase synthesis and high-resolution NMR studies of two synthetic double-helical RNA dodecamers: r(CGCGAAUUCGCG) and r(CGCGUAUACGCG)

Ten-micromole solid-phase RNA synthesis has been successfully performed on an automated nucleic acid synthesizer with coupling efficiencies up to 99%, using the tert-butyldimethylsilyl group to protect the 2'-hydroxyl. The tert-butyldimethylsilyl group was easily removed by tetrabutylammonium fluoride under conditions in which virtually no 2'- to 3'-isomerization was found to occur. By use of this approach, the self-complementary RNA dodecamers r(CGCGAAUUCGCG) and r(CGCGUAUACGCG) were synthesized on an automated nucleic acid synthesizer, purified by TLC, and studied by high-resolution NMR. Imino protons were assigned from one-dimensional nuclear Overhauser effects. The nonexchangeable base, H1', and H2' protons were assigned by the sequential NOESY connectivity method. The NOE data from these two oligomers were analyzed qualitatively and compared to the ideal A- and B-type helix models of Arnott et al. (1972a,b). The internucleotide H6/H8 NOEs to the preceding H1' in r(CGCGUAUACGCG) were found to be sequence-dependent and probably reflect the roll angles between adjacent bases. The internucleotide H6/H8 to H2' NOEs of these oligomers correspond very well to an A-type conformation, but the interstrand adenine H2 NOEs to the following H1' were much stronger than those predicted from the fiber model. These srong interstrand NOEs can be rationalized by base pair slide to favor more interstrand base overlap, as predicted by Callidine and Drew (1984).     

A multinuclear, high-resolution NMR study of bovine casein micelles and submicelles

High-resolution, natural abundance 13C[1H] (100.5 MHz), 31P[1H] (161.8 MHz) and 1H (400.0 MHz) NMR spectroscopy was used to identify the calcium-binding sites of bovine casein and to ascertain the dynamic state of amino acid residues within the casein submicelles (in 125 mM KCl, pD = 7.4) and micelles (in 15 mM CaCl2/80 mM KCl, pD = 7.2). The presence of numerous, well-resolved peaks in the tentatively assigned 13C-NMR spectra of submicelles (90 A radius) and micelles (500 A radius) suggests considerable segmental motion of both side chain and backbone carbons. The partly resolved 31P-NMR spectra concur with this. Upon Ca2+ addition, the phosphoserine beta CH2 resonance (65.8 ppm vs DSS) shifts upfield by 0.2 ppm and is broadened almost beyond detection; a general upfield shift (up to 0.3 ppm) is also observed for the 31P-NMR peaks. The T1 values of the alpha CH envelope for submicelles and micelles are essentially identical corresponding to a correlation time of 8 ns for isotropic rotation of the caseins. Significant changes in the 31P T1 values accompany micelle formation. Data are consistent with a loose and mobile casein structure, with phosphoserines being the predominant calcium-binding sites.     

31P-NMR and ESR studies of the oxidation states of manganese in Staphylococcus aureus

High-resolution 31P-NMR and ESR spectroscopies are used to probe the role of manganese in oxygen metabolism, in vivo, by Staphylococcus aureus. The linewidth of the intracellular orthophosphate resonance in the 31P-NMR spectrum and the amplitude of the ESR sextet of signals due to Mn2+ hexaquo ions are found to be sensitive to the oxygenation state of the cells. These results are attributed to changes in the oxidation state of the manganese. It is concluded that manganous ions are oxidized to Mn3+ in oxygenated cells. Mn3+ is in turn reduced to Mn2+ under anaerobic conditions. The Mn2+ is also oxidized to Mn3+ by hydrogen peroxide probably as a result of the disproportionation of H2O2 to H2O and O2 by an active catalase in S. aureus. Addition of mercaptoethanol to a suspension of oxygenated cells results in the reduction of Mn3+ to Mn2+.     

Synthesis, modification with N-acetoxy-2-acetylaminofluorene and physicochemical studies of DNA model compound d(TACGTA)

The deoxyhexanucleotide d(TACGTA) was synthesized by a modified phosphotriester method. The modified procedure made rapid synthesis of deoxyoligonucleotide possible in gram quantity. N-Acetoxy-2-acetylaminofluorene (AAAF) modified d(TACGTA). Thin layer chromatography and UV analysis of the acid treated AAF modified hexanucleotide showed that the covalent modification with AAF took place exclusively at C(8) of guanine in d(TACGTA). d(TACGTA) and AAF modified d(TACGTA) were purified by preparative high performance liquid chromatography (HPLC). The pure products were characterized by 1H and 31P-NMR. The circular dichroism (CD) spectrum of d(TACGTA) was consistent with DNA in the B form even in the presence of 4 M NaCl whereas the modified hexamer had nearly inverted spectrum in the absence of any added salt. Both NMR and CD analyses indicated profound alteration of conformation of d(TACGTA) upon covalent modification with AAF. The stabilization of the Z-like conformation in the modified hexamer under physiological conditions of salt and temperature suggests biological relevance.     

Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [2H]HEP and [2H]C4S increase substantially upon binding to LDL, with the [2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C2H3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32-35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (less than or equal to 1 mg/ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.     

NMR Study of Complexation of Aromatic Ligands with Heptadeoxynucleotide 5"-d(GCGAAGC) Forming Stable Hairpin Structure in Aqueous Solution

Complex formation of hairpin-producing heptadeoxynucleotide 5"-d(GCGAAGC) with aromatic molecules: acridine dye proflavine and anthracycline antibiotic daunomycin was studied by one-dimensional ¹H NMR and two-dimensional correlation ¹H–¹H (2D-TOCSY, 2D-NOESY), ¹H–³¹P (2D-HMBC) NMR spectroscopy (500 and 600 MHz) in aqueous solution. Concentration and temperature dependences for the chemical shifts of ligand protons were measured, molecular models of equilibrium in solution were developed, and equilibrium thermodynamic parameters for the formation of intercalation complexes were calculated. Spatial structures of dye and antibiotic complexes with the heptamer hairpin were constructed on the basis of 2D-NOE data and the calculated values of limiting chemical shifts of ligand protons.     

Interactions of ubiquinones with membrane models

The effect of the insertion of coenzyme Q10 and some of its shorter chain homologues in membrane models (Reverse Micelles, Small Unilamellar Vesicles and Liposomes) has been studied by NMR and IR spectroscopies. By 1H-NMR we have found that the stretched conformation of the isoprenoid side-chain observed in solution is maintained in membrane models. Interaction between the quinonoid moiety of the Q's and the phosphatidic groups of the phospholipids has been evidenced by 31P-NMR. A large effect of this interaction on the water microdynamics and distribution around the charged groups of the phospholipids has been observed by measurements of 1H and 2H relaxation times and by infrared spectra. The 13C-NMR spectra of the backbone of the acyl chains of phospholipids does not seem to be influenced to a noticeable extent by the presence of the Q's.     

Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA.

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.     

Interaction of a novel antitumor agent TAS-103 with DNA.

Interaction of a novel antitumor agent TAS-103 with DNA has been studied by a variety of methods including thermal melting study, UV-Visible spectroscopy, 1H- and 31P-NMR spectroscopy. Thermal melting study indicated that TAS-103 stabilizes the double stranded form of DNA and the relative binding strength of TAS-103 is equal to that of ethidium bromide (EtBr). UV-Visible spectroscopy demonstrated that titration curves are nearly identical with all DNA oligomers producing a hypochromic and hypsochromic effect. A hypsochromic effect of TAS-103 is differ from typical intercalators such as EtBr and Actinomycin D that exhibit a bathochromic effect. 1H- and 31P-NMR spectroscopy revealed that TAS-103 has mainly two binding modes. Major binding mode is outside binding and minor binding mode is intercalation.