Two-dimensional Nuclear Overhauser Effect Spectra Research Articles

Photoresponsive Behavior of Wormlike Micelles Constructed by Gemini Surfactant 12-3-12·2Br- and Different Cinnamate Derivatives.

The photoresponsive wormlike micelles constructed by Gemini surfactants and cinnamate derivatives play a great role in the field of smart materials. However, how the structure of cinnamate derivatives affects the photoresponsive behavior of micelles is still a hotspot for scientists to research. Here, three kinds of aromatic salts with different ortho-substituted groups including trans- o-methoxy cinnamate ( trans-OMCA), trans- o-hydroxy cinnamate ( trans-OHCA), and trans-cinnamate ( trans-CA) were introduced into Gemini surfactant 12-3-12·2Br- aqueous solutions to construct photoresponsive wormlike micelles through their noncovalent interactions. Their properties were researched using the rheological method, cryo-transmission electron microscopy, and 1H NMR and two-dimensional nuclear Overhauser effect spectra. The results show that these cinnamate derivatives could well construct wormlike micelles with 12-3-12·2Br-. Furthermore, subtle differences in the ortho substituents' structure have a significant effect on the photoresponsive behavior of formed wormlike micelles. Specifically, the zero viscosity (η0) of 40 mM 12-3-12·2Br-/24 mM trans-OHCA mixed solution decreases from 26.72 to 2.6 Pa·s with the shortening of the length of wormlike micelles after UV irradiation. Correspondingly, the η0 for the same ratio of 12-3-12·2Br-/ trans-OMCA decreases from 2.42 to 0.06 Pa·s and the wormlike micelles are transited into rodlike micelles and even spherical micelles after the same UV irradiation time. However, the variation of wormlike micelles in the 12-3-12·2Br-/ trans-CA system induced by UV light is not obvious with η0 being maintained at around 2.89 Pa·s. This study will help us better understand the effects of chemical groups on macrophenomena and microinteraction for micellar systems. It provides a theoretical basis for the construction of photoresponsive micelles, thus widening their application in the field of soft materials.

Conformations of Oxidized Glutathione in Aqueous Urea Solution by All-Atom Molecular Dynamic Simulations and 2D-NOESY Spectrum

All-atom molecular simulations and two-dimensional nuclear overhauser effect spectra have been used to study the conformations and interactions of oxidized glutathione (GSSG) in aqueous urea solution. The simulations were characterized by intramolecular distance, radius of gyration, solvent-accessible surface area, and root-mean-square deviation. Interestingly, the two chains connected by the GSSG disulfide linkage exhibited different flexibilities in the aqueous urea solution. GSSG can convert from “extended” to “folded” states in the simulations. Its preferred conformation in aqueous urea solutions is “extended”, which was confirmed by the 2D nuclear magnetic resonance (NMR) experiment. The two different types of amide hydrogen atoms in cysteine and glycine also showed different capacities to form N–H⋯O hydrogen bonds. The results were confirmed by temperature-dependent NMR experiment.

Conformations of Carnosine in Aqueous Solutions by All-Atom Molecular Dynamics Simulations and 2D-NOSEY Spectrum

All-atom molecular simulations and two-dimensional nuclear overhauser effect spectrum have been used to study the conformations of carnosine in aqueous solution. Intramolecular distances, root-mean-square deviation, radius of gyration, and solvent-accessible surface are used to characterize the properties of the carnosine. Carnosine can shift between extended and folded states, but exists mostly in extended state in water. Its preference for extension in pure water has been proven by the 2D nuclear magnetic resonance (NMR) experiment. The NMR experimental results are consistent with the molecular dynamics simulations.

Hidden in plain sight: subtle effects of the 8-oxoguanine lesion on the structure, dynamics, and thermodynamics of a 15-base pair oligodeoxynucleotide duplex.

The base lesion 8-oxoguanine is formed readily by oxidation of DNA, potentially leading to G → T transversion mutations. Despite the apparent similarity of 8-oxoguanine-cytosine base pairs to normal guanine-cytosine base pairs, cellular base excision repair systems effectively recognize the lesion base. Here we apply several techniques to examine a single 8-oxoguanine lesion at the center of a nonpalindromic 15-mer duplex oligonucleotide in an effort to determine what, if anything, distinguishes an 8-oxoguanine-cytosine (8oxoG-C) base pair from a normal base pair. The lesion duplex is globally almost indistinguishable from the unmodified parent duplex using circular dichroism spectroscopy and ultraviolet melting thermodynamics. The DNA mismatch-detecting photocleavage agent Rh(bpy)(2)chrysi(3+) cleaves only weakly and nonspecifically, revealing that the 8oxoG-C pair is locally stable at the level of the individual base pairs. Nuclear magnetic resonance spectra are also consistent with a well-conserved B-form duplex structure. In the two-dimensional nuclear Overhauser effect spectra, base-sugar and imino-imino cross-peaks are strikingly similar between parent and lesion duplexes. Changes in chemical shift due to the 8oxoG lesion are localized to its complementary cytosine and to the 2-3 bp immediately flanking the lesion on the lesion strand. Residues further removed from the lesion are shown to be unperturbed by its presence. Notably, imino exchange experiments indicate that the 8-oxoguanine-cytosine pair is strong and stable, with an apparent equilibrium constant for opening equal to that of other internal guanine-cytosine base pairs, on the order of 10(-6). This collection of experiments shows that the 8-oxoguanine-cytosine base pair is incredibly stable and similar to the native pair.

Solution Structure and Stability against Digestion of rproBnIb, a Recombinant 2S Albumin from Rapeseed: Relationship to Its Allergenic Properties,

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.

Structure of DNA Hexamer Sequence d-CGATCG by Two-dimensional Nuclear Magnetic Resonance Spectroscopy and Restrained Molecular Dynamics

Solution conformation of self-complementary DNA duplex d-CGATCG, containing 5′d- CpG 3′ site for intercalation of anticancer drug, daunomycin and adriamycin, has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Complete resonance assignments of all the protons (except some H5′/H5” protons) have been obtained following standard procedures based on double quantum filtered correlation spectroscopy (dQF COSY) and two-dimensional nuclear Overhauser effect (NOE) spectra. Analysis of sums of coupling constants in one-dimensional NMR spectra, cross peak patterns in dQF COSY spectra and inter proton distances shows that the DNA sequence assumes a conformation close to the B-DNA family. The deoxyribose sugar conformation is in dynamic equilibrium with predominantly S-type conformer and a minor N-type conformer with N↔S equilibrium varying with temperature. At 325 K, the mole fraction of the N-conformer increases for some of the residues by ∼ 9%. Using a total of 10 spin-spin coupling constants and 112 NOE intensities, structural refinement has been carried out using Restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols. It is observed that pseudorotation phase angle of deoxyribose sugar for A3 and T4 residues is ∼ 180° and ∼ 120°, respectively while all other residues are close to C2′endo-conformation. A large propeller twist (∼ −18°) and smallest twist angle (∼ 31°) at A3pT4 step, in the middle of the sequence, a wider (12 Å) and shallower (3.0 Å) major groove with glycosidic bond rotation as high anti at both the ends of hexanucleotide are observed. The structure shows base-sequence dependent variations and hence strong local structural heterogeneity, which may have implications in ligand binding.

High-Resolution NMR of an Antisense DNA·RNA Hybrid Containing Alternating Chirally Pure Rp Methylphosphonates in the DNA Backbone

A high-resolution proton NMR study has been performed on a hybrid duplex formed by a methylphosphonate (MP) oligodeoxyribonucleotide (MPO) and its target oligoribonucleotide, d(T(MP)CC(MP)-TT(MP)AG(MP)CT(MP)CC(MP)TG) x r(CAGGAGCUAAGGA), where MP corresponds to positions of methylphosphonate linkages in the pure R(p) stereoconfiguration. MP-containing analogs of DNA are reported to be effective antisense agents capable of specifically inhibiting protein synthesis with the R(p) chiral MPOs exhibiting greater affinity for the target mRNA than their S(p) counterparts. Nearly complete proton resonance assignments of the hybrid duplex have been made using two-dimensional nuclear Overhauser effect (2D NOE) spectra, at three different mixing times, and double quantum-filtered COSY (2QF-COSY) spectra. The 2QF-COSY cross-peak patterns which are resolved have been analyzed qualitatively to suggest sugar conformations. Distance restraints have been obtained from the 2D NOE spectra of the duplex in D(2)O. These interproton distance restraints were determined using a complete relaxation matrix method to improve accuracy. Specifically, a new approach termed RANDMARDI has been utilized to calculate these distance restraints, accounting for spectral noise and errors in 2D NOE peak volume integration. The calculated interproton distances and sugar puckers have been analyzed to assess the solution conformation of the hybrid. The hybrid duplex appears to have an overall solution structure which is distinct from standard B- and A-forms, but the RNA strand exhibits features of the A-form. The absence of H1'-H2' cross-peaks in the 2QF-COSY spectrum indicates a C3'-endo type of conformation for ribose sugars in the RNA strand. The deoxyriboses in the antisense DNA strand exhibit a mixed behavior with almost equal scalar coupling constant values for H1'-H2' and H1'-H2" and a strong H3'-H4' 2QF-COSY peak pattern. Variations in calculated values of interproton distances and sixth-root R factor analysis of experimental intensities indicate that the hybrid duplex may have a DNA strand with significant conformational plasticity.

Complete 1H and 13C resonance assignments of a 21-amino acid glycopeptide prepared from human serum transferrin

A 21-amino acid glycopeptide (Gp21) was isolated and purified in multi-milligram yields from commercially available human serum transferrin (HSTF) by a combination of tryptic digestion, Con A affinity chromatography, and reverse phase HPLC. The peptide chain of Gp21 contains a single N-glycosylation site to which a diantennary oligosaccharide is attached. The amino acid sequence and the glycan primary structure of Gp21 have been verified by peptide sequencing, electrospray mass spectrometry, and one-dimensional 1H NMR spectroscopy. Different glycoforms were found for the glycan of Gp21 derived from two different batches of commercial HSTF. These glycoforms differ from one another in the number of NeuAc residues (ranging from 0 to 2) and/or the number of Gal residues (ranging from 1 to 2). As for the monogalacto species, in the two-dimensional nuclear Overhauser effect (NOE) spectrum of Gp21, interglycosidic NOEs were observed between Man4 in the α(1 → 3) branch and the terminal GlcNAc β(1 → 2) residue. No interglycosidic NOE was observed between Man4′ in the α(1 → 6) branch and the terminal GlcNAc residue. These observations indicate that the terminal GlcNAc residue in the minor glycoforms of Gp21 is exclusively located in the α(1 → 3) branch of the Gp21 glycan. The occurrence of such a carbohydrate structure in HSTF has not been reported before. The 1H and 13C NMR spectra of Gp21 have been completely assigned by two-dimensional homonuclear and heteronuclear spectroscopy. The close similarity of the 1H and 13C chemical shift values for the Gp21 glycan with the respective values for the peptide-free diantennary oligosaccharide (Wieruszeski et al., Glycoconjugate J., 6 (1989) 183–194) indicates that the 1H and 13C chemical shifts of the diantennary oligosaccharide are not perturbed by the presence of the Gp21 peptide fragment. The complete 1H and 13C resonance assignments and the full characterization of the primary structure of Gp21 will permit us to study the conformation and dynamics of the N-linked diantennary oligosaccharides while covalently attached to a polypeptide fragment.

Parallel processing of chemical information in a local area network—III. Using genetic algorithms for conformational analysis of biomacromolecules

Multi-dimensional nuclear magnetic resonance experiments are an excellent means of revealing the three-dimensional structure of biomacromolecules in solution. However, the search space in the conformational analysis of biomacromolecules, using multi-dimensional NMR data, is huge and complex. This calls for global optimization techniques with good sampling properties. This paper describes a genetic algorithm that optimizes the fit between (simulated) experimental two-dimensional Nuclear Overhauser Effect spectra and the corresponding calculated spectra for trial structures. This is a very computational intensive procedure. Speed-up of performance is achieved by parallelizing the algorithm, i.e. creating small subpopulations of trial structures, each of which can be processed on different processors. Good sampling behavior is obtained by initializing each subpopulation with its own random seed and the introduction of a migration operator. The latter replaces the best performing individual from one subpopulation with the worst performing individual from another subpopulation after a predetermined number of generations. A parallel genetic algorithm for the conformational analysis of nucleic acids is developed using the software package HYDRA. It is demonstrated that, for the data sets used in the study, a considerable reduction in computation time is obtained for the parallel genetic algorithm as compared to a sequential implementation, while the same optimal solutions are found.

Aqueous solution structure of a hybrid lentiviral Tat peptide and a model of its interaction with HIV-1 TAR RNA.

Human immunodeficiency virus, type 1, (HIV-1) encodes a transactivating regulatory protein, called Tat, which is required for efficient transcription of the viral genome. Tat acts by binding to a specific RNA stem-loop element, called TAR, on nascent viral transcripts. The specificity of binding is principally determined by residues in a short, highly basic domain of Tat. The structure in aqueous solution of a biologically active peptide, comprised of the ten-amino acid HIV-1 Tat basic domain linked to a 15-amino acid segment of the core regulatory domain of another lentiviral Tat, i.e., that from equine infectious anemia virus (EIAV), has been determined. The restraint data set includes interproton distance bounds determined from two-dimensional nuclear Overhauser effect (2D NOE) spectra via a complete relaxation matrix analysis. Thirty structures consistent with the experimental data were generated via the distance geometry program DIANA. Subsequent restrained molecular mechanics calculations were used to define the conformational space subtended by the peptide. A large fraction of the 25-mer peptide assumes a structure in aqueous solution with the lysine- and arginine-rich HIV-1 basic domain being separated from the basic domain by a turn and characterized by a nascent helix as well. The Tat peptide/TAR complex could be modeled with the basic alpha-helix lying in the major groove of TAR such that important interactions of a putative specificity-endowing arginine are maintained and very slight widening of the major groove is entailed.

Structure of chimeric duplex junctions: solution conformation of the retroviral Okazaki-like fragment r(ccca)d(AATGA).d(TCATTTGGG) from Moloney murine leukemia virus.

We have determined the solution structure of the synthetic chimeric duplex r(ccca)d(AATGA).d(TCATTTGGG) by two-dimensional NMR, distance geometry, restrained molecular dynamics, and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The chimeric strand of this duplex consists of the last four residues of the tRNA(Pro) primer for (-) strand DNA synthesis of Moloney murine leukemia virus and the first five residues of the (-) strand DNA produced by extending this primer; the complementary DNA strand corresponds to the (+) strand product from this template. The hybrid section of this chimeric duplex assumes a structure similar to that found for pure hybrid duplexes of mixed sequence, while the DNA section assumes a conformation closer to B-form DNA. There is significant distortion of the duplex at the hybrid-DNA junction which is manifested in marked changes in the helical parameters buckle, roll, and tip, changes in glycosidic torsion angles, and changes in the backbone torsion angles delta, epsilon, and zeta. The sugar conformations also undergo large changes, from heteromerous puckers in the hybrid section to a more B-form in the DNA section. Furthermore, the intrastrand phosphate separation in the chimeric strand is more typical of A-form duplexes in the RNA section but more like B-form duplexes in the DNA section. In the DNA section the minor groove width changes gradually from B-form at the periphery and approaches hybrid-like dimensions closer to the junction. The structural discontinuities act synergistically to produce a bend of 18 +/- 3 degrees at the junction. The global structure of this sequence is similar to that previously found in the chemically analogous Okazaki fragment r(gcg)d(TATACCC).d(GGGTATACGC) in solution. Such structure homology suggests a possible link between structure and function with respect to the recognition and cleavage of the junction RNA residues in both retroviral chimeras and Okazaki fragments during reverse transcription and normal DNA replication.

Calcium-Dependent Solvation of the Myristoyl Group of Recoverin

Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to function as a calcium sensor in visual signal transduction by coupling the kinetics of the recovery phase of the photoresponse to changes in the levels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed that, in the Ca(2+)-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca(2+)-bound conformation, the myristoyl group is exposed to solution. The crystal structure of Ca(2+)-bound recoverin reveals an exposed cluster of hydrophobic residues, raising the possibility that residues in this region may function as part of an intramolecular myristoyl binding site. Fluorescence spectroscopy analysis of interactions between recoverin and 1-anilinonaphthalene-8-sulfonate (ANS) shows that an increase in solvent-accessible hydrophobic surface accompanies Ca2+ binding. 1H nuclear magnetic resonance (NMR) spectra of myristoyl protons show dispersed chemical shifts in the Ca(2+)-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca(2+)-free recoverin show NOE contacts between myristoyl protons and aromatic ring protons. Tryptophan fluorescence quenching by acrylamide indicates that the myristoyl group is in proximity to a tryptophan residue only in the Ca(2+)-free conformation. These results indicate that the myristoyl group is in contact with residues in the hydrophobic cluster in Ca(2+)-free recoverin and that it is exposed to solution in the Ca(2+)-bound conformation.

Deriving Accurate Interproton Distances from ROESY Spectra with Limited Knowledge of Scalar Coupling Constants via the CARNIVAL Algorithm. An Iterative Complete-Relaxation-Matrix Approach

A method (termed CARNIVAL) for accurately determining distances from proton homonuclear rotating-frame Overhauser effect spectroscopy (ROESY) is described. The method entails an iterative calculation of the relaxation matrix using methodology introduced with the MARDIGRAS algorithm for analysis of two-dimensional nuclear Overhauser effect spectra (B. A. Borgias and T. L. James, J. Magn. Reson. 87, 475, 1990). The situation is complicated in the case of ROESY as spectral peak intensities are influenced by resonance offset and contributions from homonuclear Hartmann-Hahn (HOHAHA) transfer if the nuclear spins are related by scalar coupling. The effects of spin-locking field strength on distance determinations and the ensuing distance errors incurred when HOHAHA corrections are made with limited knowledge of scalar ( J) coupling information have been evaluated using simulated ROESY intensities with a model peptide structure. It has been demonstrated that accurate distances can be obtained with little or no explicit knowledge of the homonuclear coupling constants over a moderate range of spin-locking field strengths. The CARNIVAL algorithm has been utilized to determine distances in a decapeptide using experimental ROESY data without measured coupling constants.

The Solution Structure of the r(gcg)d(TATACCC):d(GGGTATACGC) Okazaki Fragment Contains Two Distinct Duplex Morphologies Connected by a Junction

Okazaki fragments are important intermediates in DNA replication. Chimeric duplexes that are structurally to Okazaki fragments also occur during reverse transcription of RNA retroviruses. Such duplexes consist of an RNA-DNA chimeric strand base-paired to a pure DNA strand; hence they have a hybrid duplex "left half" covalently linked to a "right half' that is pure DNA. We have determined the solution structure of the synthetic Okazaki fragment r(gcg)d(TATACGC) by means of two-dimensional NMR, restrained molecular dynamics and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The large negative x-displacement and large positive inclination in the hybrid section of the duplex are structural characteristics similar to those found in pure hybrid duplexes. However, the DNA sugar puckers and the width and depth of the minor groove in the pure DNA section are more like B-form DNA, especially beyond the junction. Thus, this Okazaki fragment duplex assumes a conformation in solution that is chimeric mixture of hybrid-form ( H-form) and B-form duplex. The co-existence of these two different conformations in a single duplex gives rise to a structural discontinuity with a bend of ∼ 18·1 (± 0·4)° at the junction between the hybrid and DNA segments that may that may be important for reverse transcriptase binding and RNase H cleavage of such molecules. Despite the fact that the solution structure is quite different from the all A -form structure reported recently for the exact same molecule in the crystalline state, a surprising number of local helical parameters were found to be quite similar to those reported for the crystal structure.

Tailored Excitation by Shaped RF Pulses. Optimization and Implementation of Excitation Containing a Notch

Design optimization and practical implementation of tailored π/2 pulses able to provide a flat null at a narrow band of resonance frequencies (several hundred hertz) with uniform excitation and minimum phase distortion of alt other resonance frequencies (±5000 Hz) are described. The self-refocusing pulses are capable of being programmed into typical commercial NMR spectrometers and possess a practical duration (1-3 ms). A conjugate gradient method is employed in shape-design optimization. For practical implementation on a 500 MHz spectrometer, the optimized pulses are approximated by a train of width-modulated DANTE pulses. Implementation of one such shaped DANTE pulse, termed a NODE-1 pulse, on a spectrometer and application to a DNA oligomer are demonstrated via a one-dimensional proton NMR spectrum of the DNA duplex in H 2O with suppression of the water proton resonance and flat excitation of all frequencies including the imino proton resonances. Further application to a two-dimensional nuclear Overhauser effect spectrum of the DNA duplex in deuterium oxide solution with null excitation at frequencies corresponding to H2′ and H2″ resonances is illustrated.

Determination of distances involving exchangeable protons from NOESY spectra of two DNA dodecamers

Two-dimensional proton nuclear Overhauser effect (NOESY) spectra were obtained as a function of mixing time for two DNA dodecamers, 5'-CGCGAATTCGCG-3' and 5'-CGCAAATTTGCG-3', in aqueous solutions. The time evolution of cross-peak volumes was quantitatively analyzed based on the approximate solution of the relaxation/exchange equation over a range of mixing times from 30 to 150 ms. Inter-proton distances, involving exchangeable protons in key locations of the structures, were calculated and compared to the distances predicted by the crystal structures. These NMR-derived distances enhance the number of constraints, and their accuracy, for determination of solution structures of the two DNA dodecamers.

Structure of a DNA : RNA Hybrid Duplex: Why RNase H Does Not Cleave Pure RNA

The solution structure of the DNA : RNA hybrid duplex d(GTCACATG) : r(caugugac) has been determined by means of two-dimensional nuclear Overhauser effect (2D-NOE) spectra, restrained molecular dynamics and full-relaxation matrix stimulation of the 2D-NOE spectra. The DNA : RNA hybrid duplex assumes neither an A-form nor a B-form structure in solution, but an intermediate heteromerous duplex structure. The sugars of the RNA strand have a normal N-type C3′- endo conformation, but the DNA strand sugars have neither N-type nor S-type conformations; instead, they have an unexpected intermediate O4′- endo conformation. The negative x -displacement, as well as the small rise and positive inclination of the base-pairs, resembles A-form morphology but the minor groove width is intermediate between that of A-form and B-form duplexes. Both the DNA and RNA strands show prominent sequence-dependent variations in their helical parameters. Combined analysis of NOE and J-coupling data indicates that the DNA sugars are not in a dynamical two-state equilibrium. The detailed three-dimensional structure of this DNA : RNA hybrid molecule leads to a proposed model for its interaction with RNase H. Several specific structural features of the enzyme complexed with the hybrid duplex appear to explain the mechanism whereby RNase H discriminates between DNA : RNA hybrid duplexes and pure RNA : RNA duplexes.

Molten-globule conformation of Arc repressor monomers determined by high-pressure 1H NMR spectroscopy.

The conformation of the pressure-dissociated monomer of Arc repressor was characterized by 1H NMR spectroscopy. The NMR spectra of the monomer under pressure (up to 5.0 kbar; 1 bar = 100 kPa) are typical of a molten globule and they are considerably different from those of the native dimer and thermally denatured monomer. The two-dimensional nuclear Overhauser effect spectra suggest that the pressure-induced molten globule retains some secondary structure. The presence of nuclear Overhauser effects in the beta-sheet region in the dissociated state suggests that the intermonomer beta-sheet (residues 8-14) in the native dimer is replaced by an intramonomer beta-sheet. Changes in one-dimensional and two-dimensional NMR spectra prior to pressure dissociation were found and suggest the existence of a "predissociated" state.

Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 5.7 residues per turn. Unique values for helical dimensions are also specified.

Structure refinement of the chromomycin dimer-DNA oligomer complex in solution

We have refined the initial docking model of the Mg(II)-co-ordinated chromomycin-d(A 2G 2C 2T 2) complex (2 drug equivalents per duplex) by a complete relaxation matrix analysis simulation of the two-dimensional nuclear Overhauser effect (NOESY) spectrum of the complex in 2H 2O solution. This relaxation matrix refined structure of the complex exhibits the following characteristics. (1) We observe an unwound and elongated duplex that exhibits characteristics distinct from the A and B-DNA family of helices at the central (G-G-C-C) · (G-G-C-C) chromomycin dimer binding and flanking sites. On the other hand sugar puckers, glycosidic torsion angles, displacement of the base-pairs from the helix axis and the minor groove width for this central tetranucleotide segment all fall within the A-family of helical parameters. (2) The chromomycin monomers are aligned in a head-to-tail orientation in the Mg(II)-co-ordinated dimer in the complex. The chromophores are aligned with a slight tilt relative to each other and make an angle of 75 ° between their planes. The C-D-E trisaccharide segments from individual monomers adopt an extended conformation that projects in opposite directions in the dimer. The divalent metal cation is co-ordinated to the O (1) carbonyl and O (9) enolate atoms of the chromophores and aligns them such that the O (9)-Mg-O (9) angle is 170 ° while all other O-Mg-O angles are in the 95(±15) ° range. (3) The sequence specificity of the chromomycin dimer for the widened and shallower (G3-G4-C5-C6) · (G3-G4-C5-C6) minor groove binding site is associated with intermolecular hydrogen bonds formed between the OH group at C (8) of the chromophore and the minor groove NH 2 group at position 2 and N (3) groups of G4 and between the O (1) oxygen of the E-sugar and the minor groove NH 2 group at position 2 of G3 in the complex. (4) Additional intermolecular interactions are primarily van der Waals contacts between anomeric and adjacent CH 2 protons on each sugar in the C-D-E trisaccharide segments of the chromomycin dimer and the minor groove surface of the DNA. These results provide insights into the induced conformational transitions required to generate a complementary match between the drug dimer and its DNA binding site on complex formation.