Myosin light chain kinase binding to plastic

Methionine-81 and/or -8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with ¹³CH₃I to produce the sulfonium ion derivatives [S-[¹³C]methylmethionine-8]glycophorin A and [S-[¹³C]methylmethionine-8 and -81]glycophorin A. ¹³C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me₄Si. A spin-lattice relaxation time of 0.4 was observed for the ¹³C-enriched methyl resonances of the sulfonium ion derivatives of Met-8 and -81, which corresponds to an effective correlation time of < 2× 10^?10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2-mercaptoethanol produces native glycophorin A which now has the ε-carbon of the methionine residue(s) 45% isotopically enriched. The ε-carbon of Met-8 was found to occur at 15.7 ppm downfield from Me₄Si whereas the ε-carbon of Met-81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me₄Si. The spin-lattice relaxation time of both resonances was found to be ～0.3 s.     

15N n.m.r. spectroscopy: 36. 1H n.m.r. and 15N n.m.r.spectroscopic investigation on the protonation of polypeptides

¹⁵N n.m.r. (9.12 MHz) spectra of acetamide, polyglycine, poly([l-alanine) and poly(l-leucine) were measured in various acidic solvents. These solvents include dichloroacetic acid (DCA), trifluoroacetic acid (TFA), methane sulphonic acid (MSA) and fluorosulphonic acid (FSA). Full protonation of both amides and polypeptides causes downfield shifts of 17–20 ppm. Furthermore, the concentration dependence of the chemical shift was measured. In solvents which cause partial protonation, decreasing concentration of amide groups may cause downfield shifts up to 8.5 ppm, while in the case of full protonation or in the absence of protonation no concentration dependence is observable. The protonation of peptide groups induces H/D-exchange of the αC proton which was monitored by ¹H n.m.r. spectroscopy. The mechanism of this H/D-exchange is discussed.     

Phosphorus nuclear magnetic resonance studies of phosphoglucomutase and its metal ion complexes.

The ³¹P nuclear magnetic resonance of the covalently bound phosphate group at the active site of phosphoglucomutase has been examined by means of Fourier transform nuclear magnetic resonance spectroscopy. At a pD of 7.9, the chemical shift of the ³¹P nucleus is 3.8 ± 0.1 ppm downfield from 85% H₃PO₄; this shift is close to that of phosphoserine (dianionic form). Proton decoupling experiments suggest that the phosphorus of the enzymic phosphate group is coupled to protons with chemical shifts similar to those of phosphoserine. In D₂O, with proton decoupling, the ratio of the longitudinal and transverse diamagnetic relaxation times in solutions of 1.6 mm phosphoenzyme yields an approximate correlation time of 10^?7s for the ³¹P nucleus of the enzyme. This is within the range of values expected for tumbling of the entire protein molecule and suggests that the covalently attached phosphate group is immobilized or “frozen” at the active site of the enzyme by means of noncovalent interactions with adjacent groups. Consistent with this, the pK_a of the enzymic phosphate is significantly lower than that of phosphoserine. Binding of the diamagnetic activator, Mg²⁺, causes little or no change in the chemical shift of the resonance of the enzymic phosphorus from pD = 5.3 to 7.6, a downfield shift (?0.5 ± 0.1 ppm) at pD = 8.6, but an upfield shift (0.8 ±0.1 ppm) for that of phosphoserine, suggesting that bound Mg²⁺ is not coordinated to the enzymic phosphate. Independent evidence against direct coordination is provided by the paramagnetic effects of Ni²⁺ bound at the active site on the relaxation rates of the enzymic phosphorus. By assessing the paramagnetic effect of bound Ni²⁺ on both the longitudinal and transverse relaxation rates of the observed resonance, and by using correlation times determined for water proton relaxation induced by the Ni²⁺ complex, a range of Ni²⁺ to phosphorus distances of 4 to 6 Å is calculated. These distances suggest a second sphere interaction between the enzyme-bound metal and the enzymic phosphate group. Bound Ni²⁺ also markedly decreases the integrated intensity of the ³¹P resonance. Although the reason for this intensity decrease is incompletely explained, the present data establish the close proximity of the bound metal ion and the active site phosphoserine on phosphoglucomutase.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

The Nature and Origin of Chemical Shift for Intracellular Water Nuclei in Artemia Cysts

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and ¹⁷O nuclei.

We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei (¹H, ²H, and ¹⁷O) is due to significant water-macromolecular interactions.

     

Carbon-13 nuclear magnetic resonance studies of polyamino acids: the helix-coil transition of poly-L-lysine

The helix-coil transition of poly-l-lysine hydrochloride ((Lys)_n) in aqueous solution has been studied by ¹³C Fourier-transform nuclear magnetic resonance spectroscopy. As reference compounds dodeca-l-lysine hydrobromide ((Lys)^?₁₂, tri-l-lysine hydrochloride ((Lys)₃), and l-lysine hydrochloride (Lys), have been also studied by the same method. It is found that ¹³C spin-lattice relaxation times t₁ of the carbonyl and the side-chain carbons decrease sharply at pD 10.2 which is the midpoint of the transition from the random-coil to the α-helix. Similarly the T₁ values of the carbonyl groups of (Lys)^?₁₂ decrease at this point in a more moderate way, while no change is observed for those of the side-chain carbons. This is interpreted in terms of the reduced α-helicity involved for (Lys)^?₁₂.The variation of ¹³C chemical shifts with pD for (Lys)_n and (Lys)^?₁₂ show the same trend:downfield shifts at higher pD. Furthermore, nonterminal and C-terminal residues of (Lys)₃ show similar behavior. Thus it is concluded that the ¹³C chemical shift changes are caused mainly by the pD changes and not by the conformational transition. Conversion from α-helix to β-structure by elevation of temperature at pD 11.2 results in narrowing and downfield shifts of the ¹³C resonances of (Lys)_n.     

Diastereomeric phosphonate ester adducts of chymotrypsin: 31P-NMR measurements

Generation of diastereomeric phosphonate ester adducts of chymotrypsin was evidenced for the first time by ³¹P NMR and spectrophotometric kinetic measurements. ³¹P NMR signals were recorded for 4-nitrophenyl 2-propyl methylphosphonate (IMN) at 32.2 ppm and for its hydrolysis product at 26.3 ppm downfield from phosphoric acid. The inhibition of α-chymotrypsin at pH > 8.0 by the faster reacting enantiomer of IMN or 2-propyl methylphosphonochloridate (IMCl), or other phosphonate ester analogs of these compounds, all caused a ～6.0 ppm downfield shift of the ³¹P signal to the 39–40 ppm region. IMN, when applied below the stoichiometric amount of chymotrypsin, under the same conditions, generated two signals, at 39.0 and at 37.4 ppm. Scans accumulated in hourly intervals showed the decomposition of both diastereomers, with approximate half-lives of 12 h at pH 8.0 and 22°C, into a species with a resonance at 35.5 ppm. The most likely reaction to account for the appearance of this new peak is the enzymic dealkylation of the isopropyl group from the covalently bound phosphonate ester. We base this conclusion mostly on the similarity of the upfield shift to the hydrolysis of phosphonate esters. Contrary to experience with phosphate ester adducts of serine proteases, no signal was detected higher than 25.0 ppm downfield from phosphoric acid for several phosphonate ester adducts of chymotrypsin and in no case did the resonance for the adduct shift further downfield in the course of the experiments. © 1993 Wiley-Liss, Inc.     

A nuclear magnetic resonance study of the diastereoisomer complexes formed between a terguride derivative and naproxen

¹H NMR (600 MHz) measurements of chemical shift changes were made in acidified (DCI) CD₃OD/D₂O 1:9 v/v equimolar solutions of (S)- and (R,S)-6-methoxy-α-methyl-2-naphthaleneacetic acid (naproxen) in the presence of 1-(3-aminopropyl)-(5R,8S,10R)-terguride (AMP-TER). The most significant bonding interactions concurring to the formation of diastereoisomer complexes are seen as chemical shifts in proximity to the positively charged nitrogen N(6)-CH₃ and of H(12), H(13), H(14) protons of the ergoline skeleton, both the adducts having an electrostatic term and different π–π stabilizing interactions. Chemical shift data exclude any contribution of the aminopropyl chain to the chiral recognition mechanism. These findings provide an experimental basis for the enantiodiscriminative process accounting for the observed chromatographic resolutions of arylcarboxylic acids on chiral stationary phases derived from AMP-TER. © 1994 Wiley-Liss, Inc.     

31P NMR Studies of the Binding of the Oligonucleotide (Ap)3A to an Oligodeoxythymidylate Covalently Linked to an Acridine Derivative

Abstract

³¹P NMR was used to study the specific interaction of an oligodeoxynucleotide containing four thymines and covalently attached to an acridine derivative through its 3-phosphate [(Tp)₄(CH₂)₅Acr] with a complementary oligoribonucleotide (Ap)₃A.³¹P-¹H and ¹H-¹H chemical shift correlation spectroscopies were jointly used to provide the assignment of the phosphorus resonances. A downfield shift of two phosphorus resonances of (Tp)₄(CH₂)₅Acr and of two phosphorus resonances of (Ap)₄A was observed upon complex formation. The assignment of the phosphorus resonances which are downfield shifted allowed us to propose a model involving an equilibrium between several 1:1 complexes where the acridine ring is intercalated between different A.T base pairs.     

P NMR studies of O-acetylserine sulfhydrylase-B from Salmonella typhimurium

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine and bisulfide to l-cysteine and acetate in bacteria and higher plants. Enteric bacteria have two isozymes of OASS, A and B, produced under aerobic and anaerobic growth conditions, respectively, with different substrate specificities. The ³¹P chemical shift of the internal and external Schiff bases of PLP in OASS-B are further downfield compared to OASS-A, suggesting a tighter binding of the cofactor in the B-isozyme. The chemical shift of the internal Schiff base (ISB) of OASS-B is 6.2 ppm, the highest value reported for the ISB of a PLP-dependent enzyme. Considering the similarity in the binding sites of the PLP cofactor for both isozymes, torsional strain of the C5-C5′ bond (O4′-C5′-C5-C4) of the Schiff base is proposed to contribute to the further downfield shift. The chemical shift of the lanthionine external Schiff base (ESB) of OASS-B is 6.0 ppm, upfield from that of unliganded OASS-B, while that of serine ESB is 6.3 ppm. Changes in chemical shift suggest the torsional strain of PLP changes as the reaction proceeds.The apoenzyme of OASS-B was prepared using hydroxylamine as the resolving reagent. Apoenzyme was reconstituted to holoenzyme by addition of PLP. Reconstitution is pseudo-first order and exhibits a final maximum recovery of 81.4%. The apoenzyme shows no visible absorbance, while the reconstituted enzyme has a UV-visible spectrum that is nearly identical to that of the holoenzyme. Steady-state fluorescence spectra gave tryptophan emission of the apoenzyme that is 3.3-fold higher than the emission of either the native or reconstituted enzyme, suggesting that PLP is a potent quencher of tryptophan emission.     

Lead-207 NMR: a novel probe for the study of calcium-binding proteins

 The high-affinity Ca²⁺–binding sites of carp (pI 4.25) and pike (pI 5.0) parvalbumins, as well as those of mammalian calmodulin (CaM) and its C-terminal tryptic half-molecule (TR₂C), were analyzed by ²⁰⁷Pb NMR spectroscopy. For the parvalbumins, two ²⁰⁷Pb signals were observed ranging in chemical shift from ≈750 to ≈1260 ppm downfield of aqueous Pb(NO₃)₂, corresponding to ²⁰⁷Pb²⁺ bound to the two high-affinity helix-loop-helix Ca²⁺–binding sites in each of these proteins. Four ²⁰⁷Pb signals, which fall in the same chemical shift window, could be discerned for CaM. Experiments on TR₂C permitted the assignment of each signal as due to ²⁰⁷Pb²⁺ occupying a helix-loop-helix site in either the N- or the C-lobe of the intact protein. ²⁰⁷Pb and ¹H NMR titration studies on CaM provided evidence that Pb²⁺ binding to all four sites occurs simultaneously, in contrast to the behavior of this protein in the presence of Ca²⁺. Titrations of the ²⁰⁷Pb²⁺–forms of CaM and TR₂C with the antipsychotic drug trifluoperazine demonstrated that drug binding to the exposed hydrophobic surfaces in CaM causes substantial conformational changes and proceeds in a sequential manner – first the C-lobe and subsequently the N-lobe. Finally, the field dependence of CaM-bound ²⁰⁷Pb signals was examined. The ²⁰⁷Pb signal linewidths exhibited a sharp dependence on the square of the external magnetic field, a trend characteristic of relaxation via chemical shift anisotropy. Relaxation studies on TR₂C demonstrated that chemical exchange also contributes to the observed linewidths. The large chemical shift dispersion observed for the ²⁰⁷Pb signals of the three proteins studied here illustrates the remarkable sensitivity of this parameter to subtle differences in the chemical environment of the protein-bound ²⁰⁷Pb nucleus. To our knowledge, the data presented in this article comprise the first ever published example of the application of ²⁰⁷Pb NMR spectroscopy to metalloproteins.     

Properties of metal-ion coordinated imidazoles: nmr and C-2 H Exchange in Co(III) Complexes

The ¹H and ¹³C nmr spectra of Co(NH₃)₅ImH³⁺ and the ¹H nmr spectra of αCotrien(ImH)₂³⁺ and βCotrien(ImH)₂³⁺ are reported. The pK_a values determined from the dependence of the chemical shift on pH are 10.0, 9.6, and 10.1, respectively. The range of the chemical shift between the acid and base forms is unusually small in the ¹H nmr, 0.5–0.7 ppm for the C-2 H and about 0.25 ppm for the C-4 H and C-5 H. In the ¹³C nmr, C-2 and C-4 have large shifts to low field and C-5 a small shift to high field on deprotonation. The C-2 proton is not exchanged with solvent ²H under acidic or basic conditions, in marked contrast to the corresponding proton in both imidazole and 1-methylimidazole. These spectroscopic and chemical properties should be useful for the direct identification of metal-ion coordinated histidines in proteins.     

Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected ¹³C and ¹⁵N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val Cα signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the α-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by ²H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.     

The crystal and molecular structure of bis(2,2′-dipyridylamine-N,N′)di(nitrato-O)cadmium(II); Solid state 113Cd NMR,components and orientation of the chemical shift tensor relative to the donor ligands

The structure of the titled compound has been determined and refined. The structure consists of isolated molecules separated by ordinary Van der Waals' distances. The Cd atom is on a crystallographic center of symmetry. The coordination polyhedron of the Cd atom is distorted octahedral with four pyridine nitrogen donors in the equatorial plane and with axial oxygen atoms from the nitrate groups. The CdO distance is 2.599(4) Å, the CdN distances are 2.310(4) and 2.316(3) Å, and the NCdN bite angle is 79.0(1)°. The solid state magic angle spinning/cross polarization ¹¹³Cd NMR isotropic chemical shift is +51.4 ppm and the components of the chemical shift tensor are: S₁₁= −92 ppm, S₃₃ = +208 ppm and S₂₂ = +39 ppm and their directions are: in the CdN₄ plane and bisecting the NCdN bite angle, perpendicular to the CdN₄ plane and the third perpendicular to the other two, respectively. This permits the assignment of contributions to the chemical shift tensor of 50 ppm from pyridine nitrogen and of −50 ppm from nitrate oxygen. From the tensor components, atomic nitrogen can be distinguished from aliphatic nitrogen donors. Crystal data: C₂₀H₁₈O₆N₈Cd; M_r = 578.8, F(000) = 580, monoclinic, P2₁/c, a = 8.591(1), b = 16.496(2), c = 7.878(1) Å, β = 95.97°, λ = 0.71073 Å, Mo Kα, V = 1110(1) ų. Z = 2, D_m = 1.73(2), D_x = 1.73 g/cm³, μ = 10.3 cm⁻¹, R_f = 0.039, 1834 reflections, 160 parameters, T ∼ 298 K. Refinement was by full matrix least-squares with anisotropic temperature factors.     

Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts

Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.     

NMR based structural studies decipher stacking of the alkaloid coralyne to terminal guanines at two different sites in parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)]4

BackgroundTelomere elongation by telomerase gets inhibited by G-quadruplex DNA found in its guanine rich region. Stabilization of G-quadruplex DNA upon ligand binding has evolved as a promising strategy to target cancer cells in which telomerase is over expressed.MethodsInteraction of anti-leukemic alkaloid, coralyne, to tetrameric parallel [d(TTGGGGT)]₄ (Ttel7), [d(TTAGGGT)]₄ (Htel7) and monomeric anti-parallel [dGGGG(TTGGGG)₃] (Ttel22) G-quadruplex DNA has been studied using Circular Dichroism (CD) spectroscopy. Titrations of coralyne with Ttel7 and Htel7 were monitored by ¹H and ³¹P NMR spectroscopy. Solution structure of coralyne-Ttel7 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by absorption, CD and ¹H NMR.Results and conclusionsBinding of coralyne to Ttel7/Htel7 induces negative CD band at 315/300 nm. A significant upfield shift in all GNH, downfield shift in T2/T7 base protons and upfield shift (1.8 ppm) in coralyne protons indicates stacking interactions. ³¹P chemical shifts and NOE contacts of G3, G6, T2, T7 protons with methoxy protons reveal proximity of coralyne to T2pG3 and G6pT7 sites. Solution structure reveals stacking of coralyne at G6pT7 and T2pG3 steps with two methoxy groups of coralyne located in the grooves along with formation of a hydrogen bond. Binding stabilizes Ttel7/Htel7 by ~ 25–35 °C in 2:1 coralyne-Ttel7/Htel7 complex.General significanceThe present study is the first report on solution structure of coralyne-Ttel7 complex showing stacking of coralyne with terminal guanine tetrads leading to significant thermal stabilization, which may be responsible for telomerase inhibition.     

Interaction of the antimalarial drug fluoroquine with DNA,tRNA, and poly(A): 19F-NMR chemical-shift and relaxation,optical absorption,and fluorescence studies

The interaction of the fluorinated antimalarial drug fluoroquine [7-fluoro-4-(diethyl-amino-1-methylbutylamino)quinoline] with DNA, tRNA, and poly(A) has been investigated by optical absorption, fluorescence, and ¹⁹F-nmr chemical-shift and relaxation methods. Optical absorption and fluorescence experiments indicate that fluoroquine binds to nucleic acids in a similar manner to that of its well-known analog chloroquine. At low drug-to-base pair ratios, binding of both drugs appears to be random. Fluoroquine and chloroquine also elevate the melting temperature (T_m) of DNA to a comparable extent. Binding of fluoroquine to DNA, tRNA, or poly(A) results in a downfield shift of about 1.5 ppm for the ¹⁹F-nmr resonance. The chemical shift of free fluoroquine depends on the isotopic composition of the solvent (D₂O vs H₂O). The solvent isotope shift is virtually eliminated by fluoroquine binding to any one of the nucleic acids. ¹⁹F-nmr relaxation experiments were carried out to measure the spin-lattice relaxation time (T₁), ¹⁹F{¹H} nuclear Overhauser effect (NOE), off-resonance intensity ratio (R), off-resonance rotating-frame spin-lattice relaxation time (T), and linewidth for fluoroquine in the nucleic acid complexes. By accounting for intramolecular proton-fluorine dipolar and chemical-shift anisotropy contributions to the fluorine relaxation, all of the relaxation parameters for the fluoroquine–DNA complex can be well described by a motional model incorporating long-range DNA bending on the order of a microsecond and an internal motion of the drug on the order of a nanosecond. Selective NOE experiments indicate that the fluorine in the drug is near the ribose protons in the RNA complexes, but not in the DNA complex. Details of the binding evidently differ for the two types of nucleic acids. This study provides the foundation for an investigation of fluoroquine in intact cells.     

NMR Studies of Tris-Intercalation: Solution Structure and Interaction of d(CTTCGCGCGAAG) with an Acridine Trimer

Abstract

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of ¹H and ³¹P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H₅', H_5”), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O_1′ endo range, those of the internal guanosines being closer to C_2′endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the ³T⁴C⁵G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3′-5′)G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

Effects of carbohydrate-structure changes on induced shifts in differential isotope-shift 13C-N.M.R.

Deuterium-induced, ¹³C-isotope shifts are shown to vary considerably from the initially predicted values calculated for ordinary pyranose and furanose sugars, when minor structural changes are introduced into the carbohydrate ring. Both substitution of C-OH groups or reduction of C-OH to CH₂ permitted the evaluation of γ effects of OD without the contribution of β-OD-induced shifting. The observed γ-shift values for these modified structures were twice as large as those previously noted. This difference is most probably due to favored salvation. Substitution of OH at C-6 led to the predicted loss of differential isotope-shift (d.i.s.) at C-6 because of its isolation from all β and γ OD groups. The ³¹P resonances of d-glucose 6-phosphate show downfield deuterium shifts. Based on d.i.s. values, new ¹³C-shift assignments are proposed for isomaltose and 2-amino-2-deoxy-α-d-glucose. A study of acidic carbohydrates has demonstrated that isotope shifts are somewhat larger for sp²-hybridized carbon atoms whose OH groups are acidic. Relaxation times for sp² carbon atoms isolated from dipolar interaction with protons were very long in D₂O relative to their relaxation time in the H₂O environment.