Conformational microheterogeneity in a DNA double helix: structure of restriction endonuclease Bam H1 recognition site

Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC)2 in solution at 19 degrees is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2'/H2", H3' reveal that the nucleotide units G2, A3 and C6 adopt (C3'-endo, chi = 200 degrees-220 degrees) conformation while G1, T4 and C5 exhibit (C2'-endo, chi = 240 degrees-260 degrees) conformation. NMR data clearly suggest that the two strands of d(GGATCC)2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC)2. The important features are: (i) G1-G2 stack, the site of cleavage, shows an alternation in sugar pucker i.e. C2'-endo, C3'-endo as in a B-A junction, (ii) G2-A3 stack adopts a mini A-DNA, both the sugars being C3'-endo, (iii) A3-T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3'-endo, C2'-endo, (iv) T4-C5 stack adopts a mini B-DNA both the sugars being C2'-endo and (v) C5-C6 stack exhibits a B-A junction with C2'-endo, C3'-endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeneity with a structural two fold, is present in the Bam H1 recognition site.     

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy and ab-initio molecular orbital calculations.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been assessed by NMR spectroscopy and pseudorotational analysis in conjunction with its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds has prompted us to establish the relationship between proton-proton and corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4) shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred (75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and South conformers with the corresponding puckering amplitude (psi m) for ddI (1), ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m = 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2 degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6 degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a psi m of 34.7 degrees. Note that these preponderant North-sugar structures (approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and ddC (4) are not reflected in the X-ray crystal structures of 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures denosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures are found to be in the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo twist form). This means that X-ray structures of ddA (2) and ddC (4) only represent the minor conformer of the overall pseudorotamer population in solution. An assumption that the structure of the pentofuranose sugar (i.e. P and psi m) participating in conformational equilibrium described by the two-state model remains unchanged at different temperatures has been experimentally validated by assessing five unknown pseudorotational parameters with eight unique observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4') for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular structures of two new anti-HIV nucleoside analogs: 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine and 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine.

The x-ray crystal structures of two new anti-HIV compounds, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (2'-F-dd-araA) and 9-(2,3-dideoxy-2-fluoro-beta-D-threo- pentofuranosyl)hypoxanthine (2'-F-dd-aral), have been determined at two temperatures. Both crystals are in the space group P2(1)2(1)2(1), and their structures were solved by direct methods. Least-squares refinement produced final R-factors of 0.027 for the 2'-F-dd-araA structure and of 0.044 for the 2'-F-dd-aral structure, respectively. The latter structure contains a two-fold disordered conformation of the sugar moiety. All three conformers (one for 2'-F-dd-araA and two for 2'-F-dd-aral) adopt an anti chi CN glycosyl torsion angle. The sugar in the 2'-F-dd-araA structure has a C2'-endo pucker conformation, whereas the sugar in the 2'-F-dd-aral structure has a mixture of C2'-endo and C3'-endo pucker conformations. When the sugar adopts the C2'-endo conformation, the torsion angle about the C4'-C5' bond is in a transgauche+ conformation. In contrast, when the sugar adopts the C3'-endo conformation, the torsion angle about the C4'-C5' bond is in a gauche(+)-gauche- conformation. The C2'-F bond distance is 1.406(3) A, similar to that found in other aliphatic C-F bonds. The results suggest that the 2'-fluoro-2',3'-dideoxyarabinosyl nucleosides do not have a strong preference for either C2'-endo or C3'-endo sugar pucker.     

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Nuclear Overhauser effect studies of the conformations of MgATP bound to the active and secondary sites of muscle pyruvate kinase

MgATP binds both at the active site (site 1) and at a secondary site (site 2) on each monomer of muscle pyruvate kinase as previously found by binding studies and by X-ray analysis. Interproton distances on MgATP bound at each site have been measured by the time-dependent nuclear Overhauser effect in the absence and presence of phosphoenolpyruvate (P-enolpyruvate), which blocks ATP binding at site 1. Interproton distances at site 2 are consistent with a single conformation of bound ATP with a high antiglycosidic torsional angle (chi = 68 +/- 10 degrees) and a C3'-endo ribose pucker (delta = 90 +/- 10 degrees). Interproton distances at site 1, determined in the absence of P-enolpyruvate by assuming the averaging of distances at both sites, cannot be fit by a single adenine-ribose conformation but require the contribution of at least three low-energy structures: 62 +/- 10% low anti (chi = 30 degrees), C3'-endo; 20 +/- 8% high anti (chi = 55 degrees), O1'-endo; and 18 +/- 8% syn (chi = 217 degrees), C2'-endo. Although a different set of ATP conformations might also have fit the interproton distances, the mixture of conformations used also fits previously determined distances from Mn2+ to the protons of ATP bound at site 1 [Sloan, D. L., & Mildvan, A. S. (1976) J. Biol. Chem. 251, 2412] and is similar to the adenine-ribose portion of free Co(NH3)4ATP, which consists of 35% low anti, 51% high anti, and 14% syn [Rosevear, P. R., Bramson, H. N., O'Brian, C., Kaiser, E. T., & Mildvan, A. S. (1983) Biochemistry 22, 3439].(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of purine nucleotides with cobalt-hexammine, cobalt-pentammine and cobalt-tetrammine cations. Evidence for the rigidity of adenosine and flexibility of guanosine and deoxyguanosine sugar conformations.

The interaction of adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP) and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) with the [Co(NH3)6]3+, [Co(NH3)5Cl]2+ and [Co(NH3)4Cl2]+ cations has been investigated in aqueous solution with metal/nucleotide ratios (r) of 1/2, 1 and 2 at neutral pH. The solid complexes have been isolated and characterized by FT-IR and 1H-NMR spectroscopy. The complexes are polymeric in nature both in the crystalline solid and aqueous solution. The binding of the cobalt-hexammine cation is indirectly (via NH3) through the N-7 and the PO3(2-) groups of the AMP and via O-6, N-7 and the PO3(2-) of the GMP and dGMP anions (outer-sphere). The cobalt-pentammine and cobalt-tetrammine bindings are through the phosphate groups (inner-sphere) and the N-7 site (outer-sphere) of these nucleotide anions. The ribose moiety shows C2'-endo/anti conformation, in the free AMP and GMP anions as well as in the cobalt-ammine-AMP complexes, whereas a mixture of teh C2'-endo/anti and C3'-endo/anti sugar puckers were observed for the Co(NH3)6-GMP, Co(NH3)5-GMP and a C3'-endo/anti conformer for the Co(NH3)4-GMP complexes. The deoxyribose showed an O4'-endo/anti conformation for the free dGMP anion and a C3'-endo/anti for the Co(NH3)6-dGMP, Co(NH3)5-dGMP and Co(NH3)4-dGMP complexes.     

Conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d (5''-GCATGC)2 complex studied in solution by 1H-n.m.r. spectroscopy.

The conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d(5'-GCATGC)2 complex have been determined from an analysis of 1H-1H vicinal coupling constants and sums of coupling constants (J1'-2',J1'-2",epsilon 1', epsilon 2' and epsilon 2") measured from one-dimensional n.m.r. spectra and from H-1'-H-2' and H-1'-H-2" cross-peaks in high-resolution phase-sensitive two-dimensional correlation spectroscopy (COSY) and double-quantum-filtered correlation spectroscopy (DQF-COSY) experiments. The value of J3'-4' has also been estimated from the magnitude of H-3'-H-4' cross-peaks in DQF-COSY spectra and H-1'-H-4' coherence transfer cross-peaks in two-dimensional homonuclear Hartman-Hahn spectroscopy (HOHAHA) spectra. The data were analysed, in terms of a dynamic equilibrium between North (C-3'-endo) and South (C-2'-endo) conformers, by using the graphical-analysis methods described by Rinkel & Altona [(1987) J. Biomol. Struct. Dyn. 4,621-649]. The data reveal that the sugars of the 2C-5G and 3A-4T base-pairs, which form the drug-intercalation site, have strikingly different properties. The deoxyribose rings of the 2C-5G base-pair are best described in terms of an equilibrium heavily weighted in favour of the C-2'-endo geometry (greater than 95% 'S'), with a phase angle, P, lying in the range 170-175 degrees and amplitude of pucker between 35 and 40 degrees, as typically found for B-DNA. For the deoxyribose rings of the 3A-4T base-pair, however, the analysis shows that, for 3A, the C-2'-endo and C3'-endo conformers are equally populated, whereas a more limited data set for the 4T nucleotide restricts the equilibrium to within 65-75% C-2'-endo. The deoxyribose rings of the 1G-6C base-pair have populations of 70-80% C-2'-endo, typical of nucleotides at the ends of a duplex. Although drug-base-pair stacking interactions are an important determinant of the enhanced duplex stability of the complex [Searle, Hall, Denny, & Wakelin (1988) Biochemistry 27, 4340-4349], the current findings make it clear that the same interactions can be associated with considerable variations in the degree of local structural dynamics at the level of the sugar puckers.     

The solution conformation of a carbocyclic analog of the Dickerson-Drew dodecamer: comparison with its own X-ray structure and that of the NMR structure of the native counterpart

The NMR conformation of a carbocyclic analog of the Dickerson-Drew dodecamer [d(CGC-GAAT*T*CGCG)]2 containing 6'-alpha-Me carbocyclic thymidines (T*) has been determined and compared with that of its X-ray structure. The solution structure of the 6'-alpha-Me carbocyclic thymidine modified duplex has also been compared with the solution structure of the corresponding unmodified Dickerson-Drew duplex solved by us under the same experimental conditions. The NMR structures have been based on 24 experimental distance and torsion constraints per residue for [d(CGCGAAT*T*CGCG)]2 (1) and on 21 constraints per residue for the natural counterpart. In general, both final NMR structures are more close to the B-type DNA. The cyclopentane moieties of the carbocyclic thymidine residues adopt C1'-exo B-DNA type puckers (the phase angles P = 136-139 degrees and the puckering amplitudes psi = 36-37 degrees) that are close to their previously published crystal C1'-exo or C2'-endo puckers. The main differences between the two NMR structures are for beta(T*8) and epsilon, xi(T*7) backbone torsions (27-50 degrees ), for basepair twist for the 7-8 and 8-9 basepair steps (5-6 degrees), tilt for the 8-9 step (7 degrees), roll for the 7-8 step (7 degrees), shift for the 7-8 step (0.9A) and slide for the 9-10 step (0.6A). The relatively small deviations of helical structure parameters lead to structural isomorphism of these duplexes in aqueous solutions (atomic RMSD = 1.0A). The difference of the minor groove widths (less than 0.7A) in the core part of the modified duplex in comparison with the native one is much smaller than the difference between the X-ray structures of these duplexes. A detailed comparison of NMR and X-ray structure parameters showed significant monotonic differences (0.9-2.5A) for all basepair slides in both duplexes. Deviations between NMR and X-ray structure parameters for the modified duplex were also found for basepair tilt of the 4-5 step (13 degrees), rolls for the 8-9 and 10-11 steps (16 and 19 degrees), twist of the 3-4 step (8 degrees) and shift of the 9-10 step (0.9A).     

Determination of the solution conformation of adenosein 2':3'-monophosphate by nuclear magnetic resonance with lanthanide probes.

The aqueous solution conformation of adenosine 2':3'-monophosphate at pH 2.5 has been determined by a nuclear magnetic resonance method utilizing lanthanide ions as shift and relaxation probes. The ribose conformation is best described as a rapid equilibrium of 2'-endo(3'-exo) and 3'-endo(2'-exo) conformations in a ratio of approximately 2 to 1. The orientation of the base relative to ribose is restricted to a narrow range about chiCN=-70 degrees.     

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.     

Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.     

Deoxyribose conformation in [d(GTATATAC)]2: evaluation of sugar pucker by simulation of double-quantum-filtered COSY cross-peaks

Exchangeable and nonexchangeable proton and phosphorus resonances (11.75 T) of [d(GTATATAC)]2 in aqueous solution were assigned by using proton two-dimensional nuclear Overhauser effect (2D NOE) spectra, homonuclear proton double-quantum-filtered COSY (2QF-COSY) spectra, proton spin-lattice relaxation time measurements, and 31P1H heteronuclear shift correlation spectra. Due to the large line widths, it was not possible to directly extract vicinal proton coupling constant values from any spectrum including ECOSY or 2QF-COSY. However, comparison of quantitative 2QF-COSY spectral simulations with experimental spectra enabled elucidation of coupling constants. The scope and limitations of this approach were explored by computation and by use of experimental data. It was found that proton line widths exhibit some variability from one residue to the next as well as from one proton to the next within a residue and the exact line width is critical to accurate evaluation of coupling constants. Experimental 2QF-COSY spectra were not consistent with a rigid deoxyribose conformation for any of the nucleotide residues. A classical two-state model, with rapid jumps between C2'-endo (pseudorotation angle P = 162 degrees) and C3'-endo (P = 9 degrees) conformations, was able to account for the spectral characteristics of terminal residue sugars: 60% C2'-endo and 40% C3'-endo. However, the 2QF-COSY cross-peaks from the -TATATA- core could be simulated only if the classical two-state model was altered such that the dominant conformer had a pseudorotation angle at 144 degrees instead of 162 degrees. In this case, the major conformer amounted to 80-85%. Alternatively, the spectral data were consistent with a three-state model in which C2'-endo and C3'-endo conformations had the largest and smallest populations, respectively, but a third conformer corresponding to C1'-exo (P = 126 degrees) was present, consistent with recent molecular dynamics calculations. This alternative yielded populations of 50% (P = 162 degrees), 35% (P = 126 degrees), and 15% (P = 9 degrees) for the -TATATA- sugars. The spectral results indicate little variation of sugar pucker between T and A. Small differences in cross-peak component intensities and characteristic spectral distortions, however, do suggest some unquantified variation. 31P1H heteronuclear chemical shift correlation spectra manifested alternating chemical shifts and coupling constants suggestive of phosphodiester backbone conformational differences between TA and AT junctions.     

Structure and conformation of 1-beta-D-ribo furanosyl pyridin-2-one-5-carboxamide: an anti-inflammatory agent

The pyrimidine nucleoside, 1-beta-D-ribofuranosyl pyridine-2-one-5-carboxamide, is an anti inflammatory agent used in the treatment of adjuvant-induced arthritis. It is the 2-one isomer of 1-beta-D-ribofuranosyl pyridine-4-one 5-carboxamide, an unusual nucleoside isolated from the urine of patients with chronic myelogenic leukemia and an important cancer marker. Crystals of 1-beta-D-ribofuranosyl pyridine-2-one-5-carboxamide are monoclinic, space group C2, with the cell dimensions a = 31.7920(13), b = 4.6872 (3), c = 16.1838(11), beta = 93.071(3) degrees , V = 2408.2(2) A(3), D(calc) = 1.496 mg/m(3) and Z = 8 (two molecules in the asymmetric unit). The structure was obtained by the application of direct methods to diffractometric data and refined to a final R value of 0.050 for 1669 reflections with I >or= 3sigma. The nucleoside exhibits an anti conformation across the glycosidic bond (chi(CN) = -15.5 degrees , -18.9 degrees ), a C3 '-endo C2 '-exo [(3)(2)T] ribose pucker and g(+) across the C(4 ')-C(5 ') exocyclic bond. The amino group of the carboxamide group is distal from the 2-one and lacks the intramolecular hydrogen bonding found in the related 2-one molecule. Nuclear magnetic resonance studies shows also an anti conformation across the glycosidic bond but the solution conformation of the furanose ring is not the same as that found in the solid state.     

The effects of monovalent cations Li+, Na+, K+, NH4+, Rb+ and Cs+ on the solid and solution structures of the nucleic acid components. Metal ion binding and sugar conformation.

The interactions of the monovalent ions Li+, Na+, K+, NH4+, Rb+ and Cs+ with adenosine-5'-monophosphoric acid (H2-AMP), guanosine-5'-monophosphoric acid (H2-GMP) and deoxyguanosine-5'-monophosphoric acid (H2-dGMP) were investigated in aqueous solution at physiological pH. The crystalline salts M2-nucleotide.nH2O, where M = Li+, Na+, K+ NH4+, Rb+ and Cs+, nucleotide = AMP, GMP and dGMP anions and n = 2-4 were isolated and characterized by Fourier Transform infrared (FTIR) and 1H-NMR spectroscopy. Spectroscopic evidence showed that these ions are in the form of M(H2O)n+ with no direct metal-nucleotide interaction, in aqueous solution. In the solid state, Li+ ions bind to the base N-7 site and the phosphate group (inner-sphere), while the NH4+ cations are in the vicinity of the N-7 position and the phosphate group, through hydrogen bonding systems. The Na-nucleotides and K-nucleotides are structurally similar. The Na+ ions bind to the phosphate group of the AMP through metal hydration shell (outer-sphere), whereas in the Na2-GMP, the hydrated metal ions bind to the base N-7 or the ribose hydroxyl groups (inner-sphere). The Na2-dGMP contains hydrated metal-carbonyl and metal-phosphate bindings (inner-sphere). The Rb+ and Cs+ ions are directly bonded to the phosphate groups and indirectly to the base moieties (via H2O). The ribose moiety shows C2'-endo/anti conformation for the free AMP acid and its alkali metal ion salts. In the free GMP acid, the ribose ring exhibits C3'-endo/anti conformer, while a C2'-endo/anti sugar pucker was found in the Na2-GMP and K2-GMP salts and a C3'-endo/anti conformation for the Li+, NH4+, Rb+ and Cs+ salts. The deoxyribose has C3'-endo/anti conformation in the free dGMP acid and O4'-endo/anti in the Na2-dGMP, K2-dGMP and a C3'-endo/anti for the Li+, NH4+, Rb+ and Cs+ salts. An equilibrium mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers was found for these metal-nucleotide salts in aqueous solution.     

Z-RNA: the solution NMR structure of r(CGCGCG).

Left-handed double-helical Z-RNA has been studied using the ribohexanucleotide pentaphosphate r(CpGpCpGpCpG). One-dimensional and two-dimensional proton nmr experiments were used to probe the structural details of the left-handed helix in concentrated sodium perchlorate solution. In 1M NaClO4 the RNA adopts the normal A-form double helix, and in 6M NaClO4 it is nearly all in the Z form. In 4M NaClO4 it exists as nearly equal parts of A form and Z form. Resonances corresponding to both A and Z form appear in the nmr spectrum, indicating that the duplex exchanges slowly between forms. Spin-spin coupling constants between protons in the ribose rings were used to determine the sugar-pucker conformations of the individual nucleotides. Quantitative nuclear Overhauser experiments were used to determine proton-proton distances within the nucleoside, and from these distances values for the glycosidic torsion angle were determined. The results show that the cytidines adopt C2'-endo sugar puckers (S type) with pseudo-rotation phase values (P) of approximately 165 degrees. The bases are in the anti conformation, with chi values of approximately -140 degrees. The internal guanosines adopt C3'-endo sugar puckers (N type) with P approximately 18 degrees, while the 3'-terminal guanosine ribose exists in an equilibrium between S- and N-type conformations. All three guanosine bases adopt the syn conformation, with chi approximately 70 degrees. The results indicate that the solution structure of Z-RNA is very similar to that of Z-DNA.     

UV irradiation of nucleic acids: formation, purification and solution conformational analysis of the '6-4 lesion' of dTpdT

Irradiation of dTpdT with 300 kJ/m2 of 254 nm produces numerous photo-products, one of which labeled dT6pd4T[1] was purified by HPLC. dT6pd4T has a UV spectrum (H20, pH 7) with lambda max = 326 nm and lambda min = 265 nm, and a P-31 NMR resonance at -3.46 ppm (normal dTpdT occurs at -4.01 ppm; TMP, 30 degrees C). 2-D COSY NMR spectra facilitated proton resonance assignments and 2-D NOESY spectra aided analysis of spatial orientation. Carbon-13 and proton-coupled P-31 NMR spectra of dT6pd4T were also obtained. These analyses indicate: C5=C6 of dT6p- is saturated and the -pd4T base is more aromatic; the dT6p- base possesses a configuration of 5R, 6S; dT6p- and -pd4T have anti-type glycosidic conformations; furanose conformation of dT6p- is mainly C3'-endo and that of -pd4T exists in a C3'-endo in equilibrium C3'-exo; exocyclic bonds gamma (C5'-C4'), beta (05'-C5') and epsilon (C3'-03') are non-classical rotamers; dihedral angle about epsilon (C3'-03') is smaller relative to dTpdT.     

Secondary structure of the hybrid poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and stereochemical modelling within the constraints of NOE data

One-dimensional nuclear Overhauser effect (NOE) in nuclear magnetic resonance spectroscopy along with stereochemically sound model building was employed to derive the structure of the hybrid poly(rA).poly(dT) in solution. Extremely strong NOE was observed at AH2' when AH8 was presaturated; strong NOEs were observed at TH2'TH2' when TH6 was presaturated; in addition the observed NOEs at TH2' and TH2' were nearly equal when TH6 was presaturated. There was no NOE transfer to AH3' from AH8 ruling out the possibility of (C-3'-endo, low anti chi approximately equal to 200 degrees to 220 degrees) conformation for the A residues. The observed NOE data suggest that the nucleotidyl units in both rA and dT strands have equivalent conformations: C-2'-endo/C-1'-exo, anti chi approximately equal to 240 degrees to 260 degrees. Such a nucleotide geometry for rA/dT is consistent with a right-handed B-DNA model for poly(rA).poly(dT) in solution in which the rA and dT strands are conformationally equivalent. Molecular models were generated for poly(rA).poly(dT) in the B-form based upon the geometrical constraints as obtained from the NOE data. Incorporation of (C-2'-endo pucker, chi congruent to 240 degrees to 260 degrees) into the classical B-form resulted in severe close contacts in the rA chain. By introducing base-displacement, tilt and twist along with concomitant changes in the backbone torsion angles, we were able to generate a B-form for the hybrid poly(rA).poly(dT) fully consistent with the observed NOE data. In the derived model the sugar pucker is C-1'-exo, a minor variant of C-2'-endo and the sugar base torsion is 243 degrees, the remaining torsion angles being: epsilon = 198 degrees, xi = 260 degrees, alpha = 286 degrees, beta = 161 degrees and gamma = 72 degrees; this structure is free of any steric compression and indicates that it is not necessary to switch to C-3'-endo pucker for rA residues in order to accommodate the 2'-OH group. The structure that we have proposed for the polynucleotide RNA-DNA hybrid in solution is in complete agreement with that proposed for a hexamer hybrid in solution from NOE data and is inconsistent with the heteronomous model proposed for the fibrous state.     

Interaction of La (III) and Tb (III) ions with purine nucleotides: evidence for metal chelation (N-7-M-PO3) and the effect of macrochelate formation on the nucleotide sugar conformation.

The interaction of the La (III) and Tb (III) ions with adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP), and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) anions with metal/nucleotide ratios of 1 and 2 has been studied in aqueous solution in acidic and neutral pHs. The solid complexes were isolated and characterized by Fourier transform ir and 1H-nmr spectroscopy. The lanthanide (III)-nucleotide complexes are polymeric in nature both in the solid and aqueous solutions. In the metal-nucleotide complexes isolated from acidic solution, the nucleotide binding is via the phosphate group (inner sphere) and an indirect metal-N-7 interaction (outer-sphere) with the adenine N-1 site protonated. In the complexes obtained from neutral solution, metal chelation through the N-7 and the PO3(2-) group is prevailing. In aqueous solution, an equilibrium between the inner and outer sphere metal-nucleotide interaction has been observed. The ribose moiety shows C2'-endo/anti pucker in the free AMP anion and in the lanthanide (III)-AMP complexes, whereas the GMP anion with C2'-endo/anti sugar conformation exhibits a mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers in the lanthanide (III)-GMP salts. The deoxyribose has O4'-endo/anti sugar pucker in the free dGMP anion and a C3'-endo/anti, in the lanthanide (III)-dGMP complexes.     

Fluorometric studies on inclusion complexation of L/D-tryptophan by beta-cyclodextrin 6-O-pyridinecarboxylates

Novel beta-cyclodextrin (beta-CD) derivatives, bearing a nicotinic or isonicotinic moiety, have been synthesized by a convenient method in 21 and 25% yields, respectively. The stability constants (K) and Gibbs free energy changes (-DeltaG degrees ) for the inclusion complexation of beta-cyclodextrin 6-O-mono(3-pyridinecarboxylate) (1), 6-O-mono(4-pyridinecarboxylate) (2), and 6-O-monobenzoate (3) with L- and D-tryptophan have been determined by spectrofluorome try in aqueous buffer solution (pH = 7.20) at 25.0 degrees C. All of the modifications dramatically enhanced the original K for beta-CD by a factor of 30-280 and interestingly switched the original enantiomer preference for L- to D-tryptophan, thus affording the inverted enantio-selectivities of K(L)/K(D) = 2.5 for beta-CD and K(D)/K(L) = 1.2-2.1 for the modified CDs 1-3. These results are discussed from the viewpoints of the size-fit and geometrical complementary relationship between the host and guest.     

NMR studies of conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them. In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates. In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template [Ferrin, L.J., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA. The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A. In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA. In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures. The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair. However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)