Dynamics in synthetic oligonucleotides. A solid-state deuterium NMR study

Solid-state 2H NMR spectroscopy was used to investigate dynamics in the [methyl-2H]thymidine-labeled oligonucleotide [d(CGCGAAT*T*CGCG)]2. Quadrupole echo line shapes, spin-lattice relaxation, and quadrupolar echo decay rates were investigated as a function of hydration W (moles of water/moles of nucleotide) between 0 and approximately 30. The amplitude of the base motion, modeled as a fast four-site libration, or diffusion in a cone, increased slightly with higher levels of hydration. A slower component of motion about the helix axis appears at W approximately 10 and increases in rate and amplitude, leading to the intermediate rate line shape observed at W approximately 21.     

Deuterium NMR investigation of backbone dynamics in the synthetic oligonucleotide [d(CGCGAATTCGCG)]2

Backbone dynamics in the [5',5"-2H2]2'-deoxythymidine labeled duplex dodecamer [d-(CGCGAAT*T*CGC)]2 have been investigated by solid-state 2H NMR. Quadrupolar echo line shapes, spin-lattice relaxation, and quadrupolar echo decay times were obtained over hydration levels ranging from W = 0.0 to 25.2 (moles of H2O/mole of nucleotide). Variation of the line shape with changing hydration level was analyzed by using models employed in previous investigations of dodecamer base and sugar dynamics. Both fast local motions and a slower helix motion were present within the oligonucleotide. The fast motion was modeled as a four-site libration whose amplitude increased with hydration level. The root mean square amplitude of this librational model was 2-6 degrees larger than the amplitude observed in either the furanose ring or base labeled material for the entire range of hydration levels investigated. The observed line shape was inconsistent with a rapid three-site trans-gauche isomerization. A slow motion about the helix axis was observed at low water levels and increased in rate and amplitude with hydration. This motional model is in agreement with previous oligonucleotide studies.     

X-ray diffraction and calorimetric study of N-lignoceryl sphingomyelin membranes.

Differential scanning calorimetry and x-ray diffraction have been used to investigate hydrated multibilayers of N-lignoceryl sphingomyelin (C24:0-SM) in the hydration range 0-75 wt % H2O. Anhydrous C24:0-SM exhibits a single endothermic transition at 81.3 degrees C (delta H = 3.6 kcal/mol). At low hydration (12.1 wt % H2O), three different endothermic transitions are observed: low-temperature transition (T1) at 39.4 degrees C (transition enthalpy (delta H1) = 2.8 kcal/mol), intermediate-temperature transition (T2) at 45.5 degrees C, and high-temperature transition (T3) at 51.3 degrees C (combined transition enthalpy (delta H2 + 3) = 5.03 kcal/mol). On increasing hydration, all three transition temperatures of C24:0-SM decrease slightly to reach limiting values of 36.7 degrees C (T1), 44.4 degrees C (T2), and 48.4 degrees C (T3) at approximately 20 wt % H2O. At 22 degrees C (below T1), x-ray diffraction of C24:0-SM at different hydration levels shows two wide-angle reflections, a sharp one at 1/4.2 A-1 and a more diffuse one at 1/4.0 A-1 together with lamellar reflections corresponding to bilayer periodicities increasing from d = 65.4 A to a limiting value of 71.1 A. Electron density profiles show a constant bilayer thickness dp-p approximately 50 A. In contrast, at 40 degrees C (between T1 and T2) a single sharp wide-angle reflection at approximately 1/4.2 A-1 is observed. The lamellar reflections correspond to a larger bilayer periodicity (increasing from d = 69.3-80.2 A) and there is some increase in dp-p (52-56 A) with hydration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deuterium relaxation and internal motion in solid Li-DNA

As part of an effort to explore the nature of the internal motion in solid polynucleotides, the spectral densities of motion J1(omega 0) and J2(2 omega 0) have been measured for oriented, partially hydrated samples of calf thymus Li-DNA deuterated in the guanine and adenine 8-positions. Both spectral densities increase with increasing hydration level, J1 is found to be 2-5 times larger than J2, and their frequency dependence appears to be omega-1 and omega-3/2, respectively. The large values of the ratio J1/J2 rule out any in-plane torsional motion as the dominant relaxation mechanism in these samples, but a drop in this ratio at high hydration levels (G13 H2O/nucleotide) may indicate increasing contributions from such torsional motion. Although a satisfactory fit to a particular motional model has yet to be achieved, our findings show that the librational motion of the C8-D bond at or below a hydration level of 10 H2O/nucleotide is approximately uniaxial, with correlation times for the motion in the range 0.2-3.0 microseconds.     

Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination

For diapause eggs of the silkworm, Bombyx mori, diapause initiation is prevented with hydrochloric acid (HCl) at around 20 h post-oviposition while diapause status is terminated with chilling around 5°C. To investigate whether hydrogen peroxide (H(2)O(2)) and catalase expression are involved in diapause initiation and termination, the concentration of H(2)O(2), relatively higher levels of catalase mRNA and activity of catalase were compared between (1) 20-h-old diapause eggs and the HCl-treated diapause eggs, and (2) 10-day-old diapause eggs and the 5°C-chilled diapause eggs. Compared to diapause eggs, the HCl-treated eggs had significantly higher H(2)O(2) concentrations (up from approximately 1-3 μmol/g fresh mass to 5-8 μmol/g fresh mass), higher relative level of catalase mRNA (up from 0 to 35.2%) and higher catalase activity (up from 2.51 units/mg protein to 4.97 units/mg protein) at 96 h post-treatment. On the other hand, the 5°C chilling resulted in significant increases of H(2)O(2) concentration (up from 0.79 μmol/g fresh mass to 5.57 μmol/g fresh mass), relative level of catalase mRNA (up from 0 to 71.4%) and catalase activity (up from 0.88 units/mg protein to 3.42 units/mg protein) within 120 days. The results obtained in this work suggest that variations of H(2)O(2) and catalase expression in Bombyx eggs are involved in diapause initiation and termination.     

Comparative 2H- and 31P-NMR study on the properties of palmitoyllysophosphatidylcholine in bilayers with gramicidin, cholesterol and dipalmitoylphosphatidylcholine

The stoichiometric palmitoyllysophosphatidylcholine (lysoPC)/gramicidin (4:1, mol/mol) lamellar complex (Killian, J.A., De Kruijff, B., Van Echteld, C.J.A., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1983) Biochim. Biophys. Acta 728, 141-144) is a useful model system to investigate the various aspects of lipid protein interactions. To study the effect of gramicidin on local order and motion of 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC) we employed 31P and 2H nuclear magnetic resonance (NMR) using selectively deuterated lysoPC's and we compared the results to those obtained for lysoPC in bilayers with cholesterol (1:1, mol/mol) and dipalmitoylphosphatidylcholine (DPPC) (1:4, mol/mol). 2H-NMR experiments on acyl chain deuterated lysoPC showed similar quadrupole splittings in the liquid crystalline state for the lysoPC/DPPC and the lysoPC/gramicidin samples. In the lysoPC/cholesterol sample an increase of the quadrupole splitting was found. T1 measurements showed that gramicidin decreases the lysoPC acyl chain motion, especially at the C12 position. In the lysoPC/cholesterol sample an increase of motion was observed as compared to lysoPC in fluid bilayers of DPPC. 31P-NMR and 2-H-NMR measurements of lysoPC, deuterated at the alpha- and beta-position of the choline moiety, indicated an increase in headgroup flexibility in all samples as compared to the parent compound DPPC. In addition, a change in headgroup conformation was observed. The alpha- and beta-segments in all samples exhibited concerted motion. It was found that also in the polar headgroup gramicidin induces a decrease of the rate of motion.     

Anion binding to neutral and positively charged lipid membranes

Aqueous anion binding to bilayer membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Only those anions that exhibit chaotropic properties showed significant binding to POPC membranes. A detailed investigation of thiocyanate binding to neutral POPC and to positively charged mixed POPC/dihexadecyldimethylammonium bromide (DHDMAB) (8:2 mol/mol) membranes revealed changes in the 2H NMR quadrupole splittings from POPC specifically deuteriated at either the alpha-segment or the beta-segment of the choline head group which were consistent with a progressive accumulation of excess negative charge at the membrane surface with increasing SCN- concentration. Both the 2H and 31P NMR spectra indicated the presence of fluid lipids in a bilayer configuration up to at least 1.0 M NaSCN with no indication of any phase separation of lipid domains. Calibration of the relationship between the change in the 2H NMR quadrupole splitting and the amount of SCN- binding provided thiocyanate binding isotherms. At a given SCN- concentration the positively charged membranes bound levels of SCN- 3 times that of the neutral membranes. The binding isotherms were analyzed by considering both the electrostatic and the chemical equilibrium contributions to SCN- binding. Electrostatic considerations were accounted for by using the Gouy-Chapman theory. For 100% POPC membranes as well as for mixed POPC/DHDMAB (8:2 mol/mol) membranes the thiocyanate binding up to concentrations of 100 mM was characterized by a partition equilibrium with an association constant of K approximately 1.4 +/- 0.3 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of water with oriented DNA in the A- and B-form conformations.

High resolution 2H nuclear magnetic resonance (NMR) was used to investigate the interaction of D2O with solid samples of uniaxially oriented Li-DNA (B-form DNA) and Na-DNA (A- and B-form DNA). At low levels of hydration, 0 approximately 4 D2O/nucleotide, the 2H spectra shows a very weak (due to short T2) broad single resonance, suggestive of unrestricted rotational diffusion of the water. At approximately 5 or more D2O/nucleotide, the Li-DNA (B-form) spectra suddenly exhibit a large doublet splitting, characteristic of partially ordered water. With increasing hydration, the general trend is a decrease of this splitting. From our analysis we show that the DNA water structure reorganizes as the DNA is progressively hydrated. The D2O interaction with Na-DNA is rather different than with Li-DNA. Below 10 D2O/nucleotide Na-DNA is normally expected to be in the A-form, and a small, or negligible splitting is observed. In the range 9-19 D2O/nucleotide, the splitting increases with increasing hydration. Above approximately 20 D2O/nucleotide Na-DNA converts entirely to the B-form and the D2O splittings are then similar to those found in Li-DNA. We show that the complex Na-DNA results obtained in the range 0-20 D2O/nucleotide are caused by a mixture of A- and B-DNA in those samples.     

Proton NMR study and conformational analysis of d(CGT), d(TCG) and d(CGTCG) in aqueous solution. The effect of a dangling thymidine and of a thymidine mismatch on DNA mini-duplexes

Proton NMR studies of d(CGT), d(TCG) and d(CGTCG) were carried out at 300 and 500 MHz. The temperature and concentration dependence of the chemical shifts of various resonances indicates duplex formation only in the cases of d(TCG) and d(CGTCG). It is concluded that d(TCG) forms a mini-duplex stabilized by a 5'-dangling thymine base. Thermodynamic parameters of the duplex-to-coil equilibrium of the d(TCG) duplex are: delta H0 = -22.3 kcal/mol and delta S0 = -70 cal/mol. K, which correspond to approximately 40% duplex formation at 0 degrees C in a 2 mM nucleotide solution. Comparison of these data with thermodynamic parameters given earlier [Borer, P.N., Dengler, B., Tinoco, I. and Uhlenbeck, O.C. (1974) J. Mol. Biol. 86, 843-853] leads to the conclusion that the dangling base stabilization observed here is approximately equivalent to the stabilization caused by one or two additional A . T base pairs. The chemical shift behaviour of various resonances in d(CGTCG) indicates duplex formation without looping out of the thymine bases. The T X T mismatch does not seem to disturb the helical structure to a large extent. Analysis of the vicinal proton-proton coupling constants of the three compounds yielded geometrical data for the sugar rings. The data are interpreted in terms of N and S pseudorotational ranges. It is shown that a distinct conformation-transmission effect is exerted by the guanosine residues in a 5'----3' direction.     

The effects of sequence context on base dynamics at TpA steps in DNA studied by NMR.

Base dynamics, heretofore observed only at TpA steps in DNA, were investigated as a function of sequence context by NMR spectroscopy. The large amplitude conformational dynamics have been previously observed in TnAn segments where n > or = 2. In order to determine whether the dynamic characteristics occur in more general sequence contexts, we examined four self-complementary DNA sequences, [d(CTTTA-NATNTAAAG)2] (where N = A, C, T, G and N = complement of N). The anomalous broadening of the TpA adenine H2 resonance which is indicative of large amplitude base motion was observed in all nine unique four nucleotide contexts. Furthermore, all the adenine H2 resonances experienced a linewidth maximum as a function of temperature, which is a characteristic of the dynamic process. Interestingly, the temperature of the linewidth maximum varied with sequence indicating that the thermodynamics of TpA base dynamics are also sequence dependent. In one example, neither a T preceding nor an A trailing the TpA step was required for base dynamics. These results show that base dynamics, heretofore observed in only a few isolated sequences, occurs at all TpA steps which are either preceded or followed by a thymine or adenine, respectively, and may be characteristic of all TpA steps in DNA notwithstanding sequence context.     

Static disorder and librational motions of the purine bases in films of oriented Li-DNA

Solid-state 2H nuclear magnetic resonance line shapes have been obtained from folded films of oriented Li-DNA molecules with the purine bases selectively labeled with deuterium at the 8-position. From line shape simulations, the static base tilts as well as the anisotropic motional amplitudes were determined as a function of hydration level and temperature. It was found that the average tilt angle of the bases is close to 0 degrees and at a hydration of ten water molecules per nucleotide the distribution width of tilt angles about this average cannot be larger than 9 degrees (standard deviation). A slightly increased distribution width is observed at low hydration levels. The motional amplitudes are hydration dependent, with the tilting motion ranging from 4 degrees for the driest, up to 15 degrees for the wettest sample, and slightly larger amplitudes are observed for the twisting motion. The amplitude of the twisting motion is unaffected by a temperature decrease down to -60 degrees C, in contrast to the tilting motion that is suppressed at low temperatures.     

Carbon-13 nuclear magnetic resonance spectroscopy of lipids: differential line broadening due to cross-correlation effects as a probe of membrane structure

We have obtained proton-coupled carbon-13 nuclear magnetic resonance (NMR) spectra of a variety of lipid-water and lipid-drug-water systems, at 11.7 T, as a function of temperature, using the "magic-angle" sample-spinning (MAS) NMR technique. The resulting spectra show a wide range of line shapes, due to interferences between dipole-dipole and dipole-chemical shielding anisotropy interactions. The differential line-broadening effects observed are particularly large for aromatic and olefinic (sp2) carbon atom sites. Coupled spectra of the tricyclic antidepressants desipramine and imipramine, in 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophases, show well-resolved doublets having different line shapes for each of the four aromatic methine groups, due to selective averaging of the four C-H dipolar interactions due to rapid motion about the director (or drug C2) axis. 2H NMR spectra of [2,4,6,8-2H4]desipramine (and imipramine) in the same 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophase exhibit quadrupole splittings of approximately 0-2 and approximately 20 kHz, indicating an approximate magic-angle orientation of the C2-2H(1H) and C8-2H(1H) vectors with respect to an axis of motional averaging, in accord with the 13C NMR results. Selective deuteration of imipramine confirms these ideas. Spectra of digalactosyl diglyceride [primarily 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl ]-3- (alpha-D-galactopyranosyl-1-6-beta-D-galactopyranosyl)-sn-glycerol]-H2O (in the L alpha phase) show a large differential line broadening for C9 but a reduced effect for C10, consistent with the results of 2H NMR of specifically 2H-labeled phospholipids [Seelig, J., & Waespe-Saracevic, N. (1978) Biochemistry 17, 3310-3315].(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of bases in hydrated [d(CGCGAATTCGCG)]2

Solid-state 2H NMR spectroscopy has been used to investigate the dynamics of a DNA oligonucleotide with a defined sequence, [d(CGCGAATTCGCG)]2, which contains the EcoRI binding site. Quadrupole echo line shapes and spin-lattice relaxation times were obtained as a function of hydration on two different deuterated samples, both in the form of the Na salt. In one sample, the C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. In the other sample, a specifically labeled thymidine (C6 deuterated) was synthetically incorporated at the seventh position (counting 5' to 3') in the sequence. The general trends for both samples were quite similar. At all levels of hydration, the data reveal the presence of a rapid, small-amplitude libration of the bases (tau c less than or equal to 1 ns, 6 degrees-10 degrees amplitude). At the higher hydration levels (80% relative humidity or higher), the results indicate the presence of a much slower motion (tau c approximately 10-100 microseconds), which at 80% relative humidity is of small amplitude (approximately 5 degrees) and at higher hydration levels may be of larger amplitude. There is no evidence for large-amplitude (greater than +/- 10 degrees) motion on a nanosecond or faster time scale under any hydration condition. The 2H NMR results were analyzed with a dynamical model which treats the oligonucleotide as a deformable filament and which can include collective torsional fluctuations. The slow motion observed at high hydration levels is attributed to the uniform twisting mode (of the entire helix).(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR structure of a parallel-stranded DNA duplex at atomic resolution

DNA dodecamers have been designed with two cytosines on each end and intervening A and T stretches, such that the oligomers have fully complementary A:T base pairs when aligned in the parallel orientation. Spectroscopic (UV, CD and IR), NMR and molecular dynamics studies have shown that oligomers having the sequences d(CCATAATTTACC) and d(CCTATTAAATCC) form a parallel-stranded duplex when dissolved at 1:1 stoichiometry in aqueous solution. This is due to the C:C⁺ clamps on either end and extensive mismatches in the antiparallel orientation. The structure is stable at neutral and acidic pH. At higher temperatures, the duplex melts into single strands in a highly cooperative fashion. All adenine, cytosine and thymine nucleotides adopt the anti conformation with respect to the glycosidic bond. The A:T base pairs form reverse Watson–Crick base pairs. The duplex shows base stacking and NOEs between the base protons T(H6)/A(H8) and the sugar protons (H1′/H2′/H2″) of the preceding nucleotide, as has been observed in antiparallel duplexes. However, no NOEs are observed between base protons H2/H6/H8 of sequential nucleotides, though such NOEs are observed between T(CH₃) and A(H8). A three-dimensional structure of the parallel-stranded duplex at atomic resolution has been obtained using molecular dynamics simulations under NMR constraints. The simulated structures have torsional angles very similar to those found in B-DNA duplexes, but the base stacking and helicoid parameters are significantly different.     

Hydrogen peroxide affects abscisic acid binding to ABAP1 in barley aleurones.

Dramatic increases in H2O2 levels have been observed following abscisic acid (ABA) treatment of plant tissues. Following ABA treatment in aleurone cells, H2O2 reached transient levels of approximately 115 micromol/L H2O2. To determine whether ABA perception was modified by such changes, the effect of H2O2 on a recently characterized ABA-binding protein (ABAP1), cloned from barley aleurone layers, was examined. ABA binding to the protein was weakened by H2O2 in a concentration-dependent manner. A concentration of 75 micromol/L H2O2 gave a 50% decline in ABA binding in a reaction following first-order kinetics, indicative of binding-site susceptibility to its microenvironment. We monitored the unfolding of ABAP1 using steady-state and time-resolved tryptophan fluorescence, while following the capacity of ABAP1 to bind ABA. ABA binding decreased by 50% following ABAP1 denaturation with 1 mol/L guanidine hydrochloride or 2 mol/L urea, while the maximum emission spectra (lambda emi) red shifted from 338 to 347 nm at 3.5 mol/L guanidine hydrochloride and 5 mol/L urea. However, only a slight blue shift of lambda emi was observed following either ABAP1 incubation with H2O2 or binding to (+)-ABA (physiologically active ABA). The equilibrium ABA dissociation rate accelerated in the presence of 250 micromol/L H2O2, with the half-time dissociation reduced to 8 min. A comparison of inactivation kinetics and conformational changes shows that inactivation of ABAP1 occurs before any noticeable conformational change. This suggests that the ABA binding site is highly responsive to its microenvironment and is situated in a region that is more flexible than the protein molecule as a whole. The results demonstrate that H2O2, generated by ABA treatment of aleurone layers, is sufficient to affect the ABA-binding capacity of ABAP1, suggesting that this may be another level of control of ABA signal transduction.     

Hydration of Escherichia coli lipids. Deuterium T1 relaxation time studies of phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine in H2O/2H2O mixtures (9:1, v/v) were investigated with 2H-NMR. Comparison of the 2H2O spin lattice relaxation time (T1) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H2O systems. Two distinct hydration regions could be discerned in the T1 relaxation time profile. (1) A minimum of 11-16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of T1 approximately equal to 90 ms. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with T1 = 500 ms and hydration water with T1 = 80-120 ms. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

C-H ... O hydrogen bonding in solutions of methylated nucleic acid base analogs as revealed by NMR

Formation and thermodynamic characteristics of C-H ... O hydrogen bonding of methylated uracils and caffeine have been studied by nmr along two lines. 1. The concentration and temperature dependencies of the PMR spectra of 1,3-dimethyluracil (m2 1,3Ura), 1,3-dimethylthymine (m2 1,3Thy), and 1,3,6-trimethyluracil (m3 1,3,6Ura) in chloroform at high concentrations of base analogs indicated the self-association of m2 1,3Ura and m2 1,3Thy via C(6)H ... O hydrogen bonding and the competitive formation of C-H ... O bonds between carbonyl oxygens and chloroform. The intermolecular interaction energy and the arrangement of molecules in the local minima of various m2 1,3Ura dimers were calculated by the method of atom-atom potentials. The deepest minimum for the m2 1,3Ura coplanar dimer corresponds to a C(6)-H ... O hydrogen-bond formation. 2. At low concentration of m2 1,3Ura and caffeine in CCl4, C(6)-H ... O bonding for m2 1,3Ura and C(8)-H ... O bonding for caffeine with oxygens of dimethyl sulfoxide (DMSO) and acetone were observed. The association constants of these complexes were obtained at different temperatures. The enthalpies delta H, of the m2 1,3Ura-DMSO, m2 1,3Ura-accetone, caffeine-DMSO, and caffeine-acetone complexes were -2 +/- 0.1 kcal/mol. The calculations showed that the deepest minimum of the caffeine-acetone coplanar complex corresponds to C(8)-H ... O bonding with energy of -3.5 kcal/mol and that of the m2 1,3Ura-acetone complexes corresponds to C(6)-H ... O bonding with energy of -3.4 kcal/mol. The approximate correction for the solvent effect provides good agreement of the experimental data with the calculations.     

Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex

High-resolution proton and phosphorus NMR studies are reported on the self-complementary d(C1-G2-T3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplex (henceforth called O6meG.T 12-mer), which contains T3.O6meG10 interactions in the interior of the helix. The imino proton of T3 is observed at 9.0 ppm, exhibits a temperature-independent chemical shift in the premelting transition range, and broadens out at the same temperature as the imino proton of the adjacent G2.C11 toward the end of the helix at pH 6.8. We observed inter base pair nuclear Overhauser effects (NOEs) between the base protons at the T3.O6meG10 modification site and the protons of flanking G2.C11 and G4.C9 base pairs, indicative of the stacking of the T3 and O6meG10 bases into the helix. Two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) studies have permitted assignment of the base and sugar H1', H2', and H2' nonexchangeable protons in the O6meG.T 12-mer duplex. The observed NOEs demonstrate an anti conformation about all the glycosidic bonds, and their directionality supports formation of a right-handed helix in solution. The observed NOEs between the T3.O6meG10 interaction and the adjacent G2.C11 and G4.C9 base pairs at the modification site exhibit small departures from patterns for a regular helix in the O6.meG.T 12-mer duplex. The phosphorus resonances exhibit a 0.5 ppm spectral dispersion indicative of an unperturbed phosphodiester backbone for the O6meG.T 12-mer duplex. We propose a model for pairing of T3 and O6meG10 at the modification site in the O6meG.T 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase

The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.     

Stoichiometry, ATP/2e values, and energy requirements for reactions catalyzed by nitrogenase from Azotobacter vinelandii.

The stoichiometry of the nitrogenase ATP-dependent H2 evolution and ecetylene reduction reactions using S2O4(2-) as an electron source was studied by various techniques. For each mole of S2O4(2-) oxidized to 2SO3(2-) by the enzyme-catalyzed reactions at 25 degrees and pH 8, 1 mol of H2 (1 mol of ethylene for acetylene reduction) and two protons are produced. Under these conditions, 4.5 mol of ATP was hydrolyzed to ADP and inorganic phosphate for each S2O4(2-) oxidized. ATP/S2O4(2-) (ATP/2e) values determined at 5 degree intervals from 10 to 35 degrees were found to go through a minimum at 20 degrees. This effect is explained in terms of possible enzyme structure modifications. Calorimetric measurements for the enzyme-catalyzed H2 evolution and acetylene reduction reactions gave deltaH values of -32.4 and -75.1 kcal/mol of S2O4(2-), respectively.