Improved detection of long-range residual dipolar couplings in weakly aligned samples by Lee–Goldburg decoupling of homonuclear dipolar truncation

Homonuclear ¹H residual dipolar couplings (RDCs) truncate the evolution of transverse ¹H magnetization of weakly aligned molecules in high-resolution NMR experiments. This leads to losses in sensitivity or resolution in experiments that require extended ¹H evolution times. Lee–Goldburg decoupling schemes have been shown to remove the effects of homonuclear dipolar couplings, while preserving chemical shift evolution in a number of solid-state NMR applications. Here, it is shown that the Lee-Goldburg sequence can be effectively incorporated into INEPT- or HMQC-type transfer schemes in liquid state weak alignment experiments in order to increase the efficiency of the magnetization transfer. The method is applied to the sensitive detection of ¹H^N–¹³C long-range RDCs in a three-dimensional HCN experiment. As compared to a conventional HCN experiment, an average sensitivity increase by a factor of 2.4 is obtained for a sample of weakly aligned protein G. This makes it possible to detect 170 long-range ¹H^N–¹³C RDCs for distances up to 4.9 Å     

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3'-processing [dinucleotide released from each 3' end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN.     

Refinement of d(GCGAAGC) hairpin structure using one- and two-bond residual dipolar couplings

The structure of the ¹³C,¹⁵N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond ¹H-¹³C, ¹H-¹⁵N, ¹³C-¹³C and ¹³C-¹⁵N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions.     

Accurate measurement of <Superscript>15</Superscript>N–<Superscript>13</Superscript>C residual dipolar couplings in nucleic acids

New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ ¹H–¹³C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H₁–C₁–N_1/9 (MQ-HCN-QJ) or H_6/8–C_6/8–N_1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and 1/(2J_NC) (attenuated spectrum). The different types of ¹⁵N–¹³C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time ¹⁵N evolution period and the offset of two frequency-selective ¹³C pulses. The methods are demonstrated for a uniformly ¹³C, ¹⁵N-enriched 24-nucleotide stem-loop RNA sequence, helix-35, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35 sample result in an estimated precision of ca. 0.07 Hz for ¹D_NC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35, refined using independently measured observables, including ¹³C–¹H, ¹³C–¹³C and ¹H–¹H dipolar couplings.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0646-2.     

Abundance of Virus-Sized Non-DNase-Digestible DNA (Coated DNA) in Eutrophic Seawater

Total DNA concentration in 0.2-μm-pore-size Nuclepore filter filtrates (<0.2-μm fraction) of Tokyo Bay water was estimated to be 9 to 19 ng/ml by an immunochemical quantification method. Almost 90% of the DNA in the <0.2-μm fraction was found in the size fractions larger than 3.0 × 10⁵ Da and 0.03 μm, and most was not susceptible to DNase digestion, that is, consisted of non-DNase-digestible DNA (coated DNA). A significant amount of DNA was obtained from the <0.2-μm fraction of the seawater by three different methods: polyethylene glycol precipitation, direct ethanol precipitation, and ultrafilter concentration. Gel electrophoresis analysis of the isolated DNAs showed that they consisted mainly of coated DNAs with a similar molecular sizes (20 to 30 kb [1.3 × 10⁷ to 2.0 × 10⁷ Da). The abundance of the ultramicron virus-sized coated DNA in natural seawater suggests that these DNA-rich particles can be attributed to marine DNA virus assemblages and that they may be a significant phosphorus reservoir in the environment.     

Solution structure of tRNA<Superscript>Val</Superscript> from refinement of homology model against residual dipolar coupling and SAXS data

A procedure is presented for refinement of a homology model of E. coli tRNA^Val, originally based on the X-ray structure of yeast tRNA^Phe, using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N–H^N RDCs measured with Pf1 phage alignment, and 20 imino N–H^N RDCs obtained from magnetic field dependent alignment of tRNA^Val. The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA^Phe by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q _free = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA^Phe, in agreement with previous NMR-based tRNA^Val models. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Validation of X-ray Crystal Structure Ensemble Representations of SARS-CoV-2 Main Protease by Solution NMR Residual Dipolar Couplings

Considerable debate has focused on whether sampling of molecular dynamics trajectories restrained by crystallographic data can be used to develop realistic ensemble models for proteins in their natural, solution state. For the SARS-CoV-2 main protease, M^pro, we evaluated agreement between solution residual dipolar couplings (RDCs) and various recently reported multi-conformer and dynamic-ensemble crystallographic models. Although Phenix-derived ensemble models showed only small improvements in crystallographic R_free, substantially improved RDC agreement over fits to a conventionally refined 1.2-Å X-ray structure was observed, in particular for residues with above average disorder in the ensemble. For a set of six lower resolution (1.55–2.19 Å) M^pro X-ray ensembles, obtained at temperatures ranging from 100 to 310 K, no significant improvement over conventional two-conformer representations was found. At the residue level, large differences in motions were observed among these ensembles, suggesting high uncertainties in the X-ray derived dynamics. Indeed, combining the six ensembles from the temperature series with the two 1.2-Å X-ray ensembles into a single 381-member “super ensemble” averaged these uncertainties and substantially improved agreement with RDCs. However, all ensembles showed excursions that were too large for the most dynamic fraction of residues. Our results suggest that further improvements to X-ray ensemble refinement are feasible, and that RDCs provide a sensitive benchmark in such endeavors. Remarkably, a weighted ensemble of 350 PDB M^pro X-ray structures provided slightly better cross-validated agreement with RDCs than any individual ensemble refinement, implying that differences in lattice confinement also limit the fit of RDCs to X-ray coordinates.     

Structure of duplex DNA containing the cisplatin 1,2-{Pt(NH3)2}-d(GpG) cross-link at 1.77 Å resolution

We report the 1.77-Å resolution X-ray crystal structure of a dodecamer DNA duplex with the sequence 5′-CCTCTGGTCTCC-3′ that has been modified to contain a single engineered 1,2-cis-{Pt(NH₃)₂}²⁺-d(GpG) cross-link, the major DNA adduct of cisplatin. These data represent a significant improvement in resolution over the previously published 2.6-Å structure. The ammine ligands in this structure are clearly resolved, leading to improved visualization of the cross-link geometry with respect to both the platinum center and to the nucleobases, which adopt a higher energy conformation. Also better resolved are the deoxyribose sugar puckers, which allow us to re-examine the global structure of platinum-modified DNA. Another new feature of this model is the location of four octahedral [Mg(H₂O)₆]²⁺ ions associated with bases in the DNA major groove and the identification of 124 ordered water molecules that participate in hydrogen-bonding interactions with either the nucleic acid or the diammineplatinum(II) moiety.     

RDC derived protein backbone resonance assignment using fragment assembly

Experimental residual dipolar couplings (RDCs) in combination with structural models have the potential for accelerating the protein backbone resonance assignment process because RDCs can be measured accurately and interpreted quantitatively. However, this application has been limited due to the need for very high-resolution structural templates. Here, we introduce a new approach to resonance assignment based on optimal agreement between the experimental and calculated RDCs from a structural template that contains all assignable residues. To overcome the inherent computational complexity of such a global search, we have adopted an efficient two-stage search algorithm and included connectivity data from conventional assignment experiments. In the first stage, a list of strings of resonances (CA-links) is generated via exhaustive searches for short segments of sequentially connected residues in a protein (local templates), and then ranked by the agreement of the experimental ¹³C_α chemical shifts and ¹⁵N-¹H RDCs to the predicted values for each local template. In the second stage, the top CA-links for different local templates in stage I are combinatorially connected to produce CA-links for all assignable residues. The resulting CA-links are ranked for resonance assignment according to their measured RDCs and predicted values from a tertiary structure. Since the final RDC ranking of CA-links includes all assignable residues and the assignment is derived from a “global minimum”, our approach is far less reliant on the quality of experimental data and structural templates. The present approach is validated with the assignments of several proteins, including a 42 kDa maltose binding protein (MBP) using RDCs and structural templates of varying quality. Since backbone resonance assignment is an essential first step for most of biomolecular NMR applications and is often a bottleneck for large systems, we expect that this new approach will improve the efficiency of the assignment process for small and medium size proteins and will extend the size limits assignable by current methods for proteins with structural models.     

Fast methionine-based solution structure determination of calcium-calmodulin complexes

Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca²⁺-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaM’s backbone conformation and a structural plasticity in CaM’s domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant. Although RDCs can define CaM domain orientation in the complex, they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs. Here we employ a specific isotope labeling strategy in which the role of methionine in CaM-peptide interactions is exploited to collect these critical NOEs. By ¹H, ¹³C-labeling the methyl groups of deuterated methionine against a ²H, ¹²C background, we can acquire a ¹³C-edited NOESY characterized by simplified, easily analyzable spectra. Together with measured CaM backbone H_N-N RDCs and intrapeptide NOE-based distances, these intermolecular NOEs provide restraints for a low temperature torsion-angle dynamics and simulated annealing protocol used to calculate the complex structure. We have applied our method to a CaM complex previously solved through X-ray crystallography: Ca²⁺-CaM bound to the CaM kinase I peptide (PDB code: 1MXE). The resulting structure has a backbone RMSD of 1.6 Å to that previously published. We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome. In addition to having application for fast complex structure determination, this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully ¹³C, ¹⁵N-labeled CaM fails.     

6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O⁶-alkylguanine DNA alkyltransferase

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate AGT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, AGT reduced the fluorescence intensity by ∼40% at binding saturation, whether the FAM was located at the 5′ or the 3′ end of the DNA. AGT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that AGT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC₅₀ ∼ 76 μM) and repair of DNA containing an O⁶-methylguanine residue (IC₅₀ ∼ 63 μM). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent AGT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy.     

[Cu4(H2O)4(dmapox)2(btc)]n · 10nH2O: The first two-dimensional polymeric copper(II) complex with bridging μ-trans-oxamidate and μ4-1,2,4,5-benzentetracarboxylato ligands: Synthesis, crystal structure and DNA binding studies

A two-dimensional copper(II) polymer with formula of [Cu₄(H₂O)₄(dmapox)₂(btc)]_n · 10nH₂O, where dmapox is the dianion of N,N′-bis[3-(dimethylamino)propyl]oxamide and btc is the tetra-anion of 1,2,4,5-benzenetetracarboxylic acid, was synthesized and characterized by elemental analysis, conductivity measurement, IR and electronic spectral studies. The crystal structure of the complex has been determined by X-ray single-crystal diffraction. The structure consists of crystallized water molecules and neutral two-dimensional copper(II) coordination polymeric networks constructed both by the bis-tridentate μ-trans-dmapox and tetra-monodentate μ₄-btc bridging ligands. Each btc ligand links four trans-dmapox-bridged binuclear copper(II) building blocks [Cu₂(H₂O)₂(trans-dmapox)]²⁺ and each binuclear copper(II) building block attaches to two btc ligands forming an infinite 2D layer which consists of 4+4 grids with dimensions of 13.563(5) × 15.616(5) Å. The environment around the copper(II) atom can be described as a distorted square-pyramid and the Cu?Cu separations through μ-trans-dmapox and μ₄-btc bridging ligands are 5.225 Å (Cu1-Cu1ⁱ), 5.270 Å (Cu2-Cu2ⁱⁱ), 6.115 Å (Cu1-Cu2), 9.047 Å (Cu1-Cu2ⁱⁱⁱ) and 10.968 Å (Cu1-Cu1ⁱⁱⁱ), respectively. Abundant hydrogen bonds among the crystallized, the coordinated water molecules, and the uncoordinated carboxyl oxygen atoms cross-link the two-dimensional layers into an overall three-dimensional channel-like framework. The interaction of the copper(II) polymer with calf thymus DNA (CT-DNA) has been investigated by using absorption, emission spectral and electrochemical techniques. The results indicate that the copper(II) polymer interacts with DNA strongly (K_b = 4.8 × 10⁵ M⁻¹ and K_sv = 1.1 × 10⁴) and the interaction mode between the copper(II) polymer and DNA may be the groove binding. To the best of our knowledge, this is the first report about the crystal structure and DNA-binding studies of a two-dimensional copper(II) polymer bridged both by the trans-oxamidate and btc ligands.     

Measurement of imino <Superscript>1</Superscript>H–<Superscript>1</Superscript>H residual dipolar couplings in RNA

Imino ¹H–¹⁵N residual dipolar couplings (RDCs) provide additional structural information that complements standard ¹H–¹H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino ¹H–¹H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA^Val using a BEST-Jcomp-HMQC2 experiment. ¹H–¹H RDCs are observed between the imino protons in G–U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino ¹H–¹H RDCs complement standard ¹H–¹⁵N RDCs because the ¹H–¹H vectors generally point along the helical axis, roughly perpendicular to ¹H–¹⁵N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the standard experiment. The ability to measure imino ¹H–¹H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

NMR studies of aurein 1.2 analogs

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA > aurein 1.2 >> the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by ¹H NMR spectroscopy, including structural refinement by natural abundance ¹³C^α, ¹³C^β, and ¹⁵N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2-5 Å below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide-lipid NOE patterns.     

Refined NMR structure of α-sarcin by 15N–1H residual dipolar couplings

¹⁵N–¹H residual dipolar couplings (RDC) have been used as additional restraints to refine the solution structure of the ribotoxin -sarcin. The RDC values were obtained by partial alignment of -sarcin in the binary mixture of n-dodecyl hexa(ethylene glycol)/hexanol. A total of 131 RDCs were measured and 106 were introduced in the final steps of the calculation protocol following the main calculation based on nuclear Overhauser enhancements and torsion angle restraints. A homogeneous family of 81 conformers was obtained. The resulting average pairwise root-mean-square deviation corresponding to the superposition of the 20 best structures is 0.69±0.12 Å for the backbone and 1.29±0.14 Å for all heavy atoms. The new structural features derived from the refined structure, compared with the non-refined structure of -sarcin, consist of new hydrogen bonds and a better definition of the backbone conformation. In particular, the loop segment spanning Gly 60 to Lys 70 shows a single conformation, corresponding to the most populated family of conformers observed in the unrefined structure. The information derived from the analysis of the refined structure and the comparison with the homologous protein restrictocin could help in establishing further structure–function relationships concerning -sarcin which can be reasonably extrapolated to other members of the ribotoxin family.     

Molecular characterization of the genomes of actinophages SH3, SH10, SH11, and SH12 infecting Streptomyces hygroscopicus.

Summary Some physico-chemical properties of the DNAs released from the actinophages SH3, SH10, SH11, and SH12 are described. The four phage DNAs have a linear double-stranded secondary structure and are unique with respect to their high G·C contents which, from melting studies and buoyant density experiments, were found to be in the range of 68–73 mol-%. The DNA molecular weights were determined by sedimentation velocity experiments and by electron microscopic length measurements, the mean values of the two corresponding data sets being 34.0·10⁶ (SH3), 26.7·10⁶ (SH10), 26.1·10⁶ (SH11), and 28.7·10⁶ (SH12) with a mean relative error of ±5%. From different observations it was concluded that SH10 DNA, and possibly also SH11 and SH12 DNA, have cohesive ends and can undergo intramolecular or intermolecular association to form ring-like monomers or linear and ring-like multimers. Cleavage of the DNAs of SH3, SH10, SH11, and SH12 by EcoRI restriction endonuclease delivered two, one, zero, and two cleavage sites, respectively, and by BamHI restriction endonuclease eight, zero, zero, and zero cleavage sites, respectively.     

5-Methylcytosine content and methylation status in six millet DNAs

High performance liquid chromatographic analysis of the total nuclear DNAs of 6 millets plant species indicates that the 5-methylcytosine content ranges from 3% in barn yard millet to 9.6% in great millet while the fraction of cytosines methylated varies between 14% in little millet to 31 % in pearl millet. Digestion of millet DNAs with MspI/HpaII suggests that CpG methylation is more in great millet DNA while CpC methylation is more in the other 5 millet DNAs. Digestion of millet DNAs with MboI, Sau3AI andDpnI indicates that some of the^5’ GATC^3’ sequences are methylated at adenine and/or cytosine residues except in little millet where adenine methylation of the^5’GATC^3’ sequences is insignificant and there is a predominance of cytosine methylation in these sequences.     

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: Comparison of unrefined and refined structure sets with the crystal structure

The assignment of the ¹H and ¹⁵Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain -spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.