Refinement of the protein backbone angle ψ in NMR structure calculations

Cross-correlated relaxation rates involving the C-H dipolar interaction and the carbonyl (C) chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle can be directly refined against such cross-correlated relaxation rates (^{H C,C}) and the three-bond H/D isotope effect on the C chemical shifts (³C _(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle is defined. We have applied the new refinement method to the -Spectrin SH3 domain (a -sheet protein) and to the Sgs1p HRDC domain (an -helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the -refined NMR structures of the SH3 domain deviate less from the 1.8 Å crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.     

Does water play a structural role in the folding of small nucleic acids?

Nucleic acid structure and dynamics are known to be closely coupled to local environmental conditions and, in particular, to the ionic character of the solvent. Here we consider what role the discrete properties of water and ions play in the collapse and folding of small nucleic acids. We study the folding of an experimentally well-characterized RNA hairpin-loop motif (sequence 5'-GGGC[GCAA]GCCU-3') via ensemble molecular dynamics simulation and, with nearly 500 micros of aggregate simulation time using an explicit representation of the ionic solvent, report successful ensemble folding simulations with a predicted folding time of 8.8(+/-2.0) micros, in agreement with experimental measurements of approximately 10 micros. Comparing our results to previous folding simulations using the GB/SA continuum solvent model shows that accounting for water-mediated interactions is necessary to accurately characterize the free energy surface and stochastic nature of folding. The formation of the secondary structure appears to be more rapid than the fastest ionic degrees of freedom, and counterions do not participate discretely in observed folding events. We find that hydrophobic collapse follows a predominantly expulsive mechanism in which a diffusion-search of early structural compaction is followed by the final formation of native structure that occurs in tandem with solvent evacuation.     

Oxidative degradation of cationic metalloporphyrins in the presence of nucleic acids: a way to binding constants?

Mn(III) and Fe(III) complexes of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (M-TMePyP) and related hybrid molecules ("metalloporphyrin-ellipticine") were activated by potassium monopersulfate in the presence of variable calf thymus (CT) DNA and NaCl concentrations. Monitored by visible spectroscopy (Soret band), fast degradation of the free metalloprophyrin was observed while the DNA-bound form appeared protected. This direct quantitation of free versus bound metalloporphyrin ratios allowed determination of binding constants: Mn- and Fe-TMePyP respectively bind to CT DNA (5 mM phosphate buffer, 0.1 M NaCl, pH 7) with K = 3 X 10(4) and 1.2 X 10(4) M-1. Mn-TMePyP showed a greater affinity for poly[d(A-T)] (K = 1.2 X 10(5) M-1) than for poly[d(G-C)] (K = 0.2 X 10(4) M-1). This method allowed us access to the intrinsic DNA affinity of the metalloporphyrin moiety of the hybrid molecules "metalloporphyrin-ellipticine".     

Novel three-dimensional 1H−13C−31P triple resonance experiments for sequential backbone correlations in nucleic acids

Summary Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, ¹H–¹³C–³¹P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of ¹H–³¹P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihydral angle constraints. By relying on ¹H–³¹P rather than ¹³C–³¹P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of ¹H–³¹P and ¹³C–³¹P couplings provide dihedral angle constraints for RNA structure determination.     

Effects ofγ-irradiation on embryonic development and nucleic acids in trigeminal neurons

Summary Pregnant rats were exposed to absorbed doses of 0.5, 1, 2, and 4 Gy of⁶⁰Co- rays either on day 8 or on day 12 p.c.. The embryos were collected on day 18 p.c.. Irradiations both on day 8 and 12 p.c. result in a dose dependent decrement of weight. The effect is larger for irradiation on day 8 p.c.. Caryometric studies of the neurons in the trigeminal ganglion show, on the other hand, that the reduction of nuclear sizes in this tissue is more substantial for an irradiation on day 12 p.c. when the ganglion is in a stage of formation. Cytophotometric determinations of the Feulgen-DNA content and determination of the RNA content by staining with chromic gallocyanin in combination with ribonuclease digestion lead to the conclusion that the irradiation induces no significant hyperploidy and that the irradiated neurons have the nuclear RNA content that is normal at this stage of gestation. This applies both to the irradiations on day 8 and on day 12 p.c.     

Extraction of total nucleic acids from bacterial isolates using the bioMérieux NucliSENS easyMAG total nucleic acid extractor

The BioMerieux NucliSENS easyMAG total nucleic acid extractor was evaluated for use on bacterial isolates in the clinical microbiology laboratory. Forty eight isolates were extracted, yielding quantifiable amounts of DNA for all isolates. The easyMAG is appropriate for DNA extraction from bacterial isolates and will be incorporated in the clinical laboratory.     

A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein,an intrinsically disordered protein

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H^N–H^α and H^N–H^N NOEs, values for ³J_HNHα, ¹J_HαCα, ²J_CαN, and ¹J_CαN, as well as chemical shifts of ¹⁵N, ¹³C^α, and ¹³C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PP_II region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the α_R region (10–20%) is found. Analysis of ¹H–¹H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.     

Pairwise NMR experiments for the determination of protein backbone dihedral angle Φ based on cross-correlated spin relaxation

Novel cross-correlated spin relaxation (CCR) experiments are described, which measure pairwise CCR rates for obtaining peptide dihedral angles Φ. The experiments utilize intra-HNCA type coherence transfer to refocus 2-bond coupling evolution and generate the or multiple quantum coherences which are required for measuring the desired CCR rates. The contribution from other coherences is also discussed and an appropriate setting of the evolution delays is presented. These CCR experiments were applied to ¹⁵N- and ¹³C-labeled human ubiquitin. The relevant CCR rates showed a high degree of correlation with the Φ angles observed in the X-ray structure. By utilizing these CCR experiments in combination with those previously established for obtaining dihedral angle Ψ, we can determine high resolution structures of peptides that bind weakly to large target molecules.     

Sequence-specific transitions of the torsion angle gamma change the polar-hydrophobic profile of the DNA grooves: implication for indirect protein–DNA recognition

Variations of the shape and polarity of the DNA grooves caused by changes of the DNA conformation play an important role in the DNA readout. Despite the fact that non-canonical trans and gauche- conformations of the DNA backbone angle γ (O5′–C5′–C4′–C3′) are frequently found in the DNA crystal structures, their possible role in the DNA recognition has not been studied systematically. In order to fill in this gap, we analyze the available high-resolution crystal structures of the naked and complexed DNA. The analysis shows that the non-canonical γ angle conformations are present both in the naked and bound DNA, more often in the bound vs. naked DNA, and in the nucleotides with the A-like vs. the B-like sugar pucker. The alternative angle γ torsions are more frequently observed in the purines with the A-like sugar pucker and in the pyrimidines with the B-like sugar conformation. The minor groove of the nucleotides with non-canonical γ angle conformation is more polar, while the major groove is more hydrophobic than in the nucleotides with the classical γ torsions due to variations in exposure of the polar and hydrophobic groups of the DNA backbone. The propensity of the nucleotides with different γ angle conformations to participate in the protein–nucleic acid contacts in the minor and major grooves is connected with their sugar pucker and sequence-specific. Our findings imply that the angle γ transitions contribute to the process of the protein–DNA recognition due to modification of the polar/hydrophobic profile of the DNA grooves.     

Quantitative Γ-HCNCH: determination of the glycosidic torsion angle χ in RNA oligonucleotides from the analysis of CH dipolar cross-correlated relaxation by solution NMR spectroscopy

A novel NMR pulse sequence is introduced to determine the glycosidic torsion angle χ in ¹³C,¹⁵N-labeled oligonucleotides. The quantitative Γ-HCNCH measures the dipolar cross-correlated relaxation rates (pyrimidines) and (purines). Cross-correlated relaxation rates of a ¹³C,¹⁵N-labeled RNA 14mer containing a cUUCGg tetraloop were determined and yielded χ-angles that agreed remarkably well with data derived from the X-ray structure of the tetraloop. In addition, the method was applied to the larger stemloop D (SLD) subdomain of the Coxsackievirus B3 cloverleaf 30mer RNA and the effect of anisotropic rotational motion was examined for this molecule. It could be shown that the χ-angle determination especially for nucleotides in the anti conformation was very accurate and the method was ideally suited to distinguish between the syn and the anti-conformation of all four types of nucleotides. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

β-PNA: peptide nucleic acid (PNA) with a chiral center at the β-position of the PNA backbone

Peptide nucleic acid (PNA) monomers with a methyl group at the β-position have been synthesized. The modified monomers were incorporated into PNA oligomers using Fmoc chemistry for solid-phase synthesis. Thermal denaturation and circular dichroism (CD) studies have shown that PNA containing the S-form monomers was well suited to form a hybrid duplex with DNA, whose stability was comparable to that of unmodified PNA–DNA duplex, whereas PNA containing the R-form monomers was not.     

Terbium(3+) as a probe of nucleic acids structure. Does it alter the DNA conformation in solution?

At low ionic strength, Tb3+ binding strongly alters the secondary structure of DNA. Circular dichroism and electro-optical techniques are more sensitive than fluorescence to study these alterations in double-stranded DNA, at low Tb3+/DNA phosphate (I/P) ratios. Both techniques yield the following conclusion: as I/P is increased, native and sonicated DNA undergo a transition from the B- to psi-form, the latter being a compact structure characteristic of aggregated DNA. Our study of alkylated DNA establishes that the accessibility of N-7 guanine to Tb3+ is clearly required for structural alterations in an aggregated state to occur. The chelation of the phosphate group and of the N-7 guanine by Tb3+ simultaneously alters the geometry of the sugar-phosphate backbone and the stacking interaction between the bases in double-stranded DNA.     

A method for the estimation of χ1 torsion angles in proteins

Summary A method for estimating CH-CH coupling constants from the shape and fine structure of NH-CH fingerprint-region cross peaks of COSY spectra is presented. Spectral simulations have been used to analyse the effect of variations in ³J_NH-CH, ³J_CH-CH, linewidths and digital resolution on the appearance of NH-CH COSY cross peaks. On the basis of these simulations a set of rules for broadly categorising experimental NH-CH cross peaks according to CH-CH coupling constants has been devised. The method has been applied to the analysis of NH-CH cross peaks of hen lysozyme. The results are compared to previous measurements of CH-CH coupling constants using E.COSY techniques.     

Determination of haloacetic acids in human urine by headspace gas chromatography–mass spectrometry

Haloacetic acids (HAAs) are water disinfection byproducts (DBPs) formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromine. HAAs are second to trihalomethanes as the most commonly detected DBPs in surface drinking water and swimming pools. After oral exposure (drinking, showering, bathing and swimming), HAAs are rapidly absorbed from the gastrointestinal tract and excreted in urine. Typical methods used to determine these compounds in urine (mainly from rodents) only deal with one or two HAAs and their sensitivity is inadequate to determine HAA levels in human urine, even those manual sample preparation protocols which are complex, costly, and neither handy nor amenable to automation. In the present communication, we report on a sensitive and straightforward method to determine the nine HAAs in human urine using static headspace (HS) coupled with GC–MS. Important parameters controlling derivatisation and HS extraction were optimised to obtain the highest sensitivity: 120 μl of dimethylsulphate and 100 μl of tetrabutylammonium hydrogen sulphate (derivatisation regents) were selected, along with an excess of Na₂SO₄ (6 g per 12 ml of urine), an oven temperature of 70 °C and an equilibration time of 20 min. The method developed renders an efficient tool for the precise and sensitive determination of the nine HAAs in human urine (RSDs ranging from 6 to 11%, whereas LODs ranged from 0.01 to 0.1 μg/l). The method was applied in the determination of HAAs in urine from swimmers in an indoor swimming pool, as well as in that of non-swimmers. HAAs were not detected in the urine samples from non-swimmers and those of volunteers before their swims; therefore, the concentrations found after exposure were directly related to the swimming activity. The amounts of MCAA, DCAA and TCAA excreted from all swimmers are related to the highest levels in the swimming pool water.     

Measurement of eight scalar and dipolar couplings for methine–methylene pairs in proteins and nucleic acids

A new 3D, spin-state-selective coherence transfer NMR experiment is described that yields accurate measurements for eight scalar or dipolar couplings within a spin system composed of a methylene adjacent to a methine group. Implementations of the experiment have been optimized for proteins and for nucleic acids. The experiments are demonstrated for C–C moieties of the third IgG-binding domain from Streptococcal Protein G (GB3) and for C –C groups in a 24-nt RNA oligomer. Chemical shifts of C, C and H (respectively C , C and H ) are dispersed in the three orthogonal dimensions, and the absence of heteronuclear decoupling leads to distinct and well-resolved E.COSY multiplet patterns. In an isotropic sample, the E.COSY displacements correspond to ¹J_CH, ²J_CH2+²J_CH3, ²J_CH, ¹J_CH2+¹J_CH3, ¹J_CH2–²J_H2H3, ¹J_CH3–²J_H2H3, ³J_HH2 and ³J_HH3 for proteins, and ¹J , ²J J , ²J , ¹J +¹J , ¹J J , ¹J J , ³J and ³J in nucleic acids. The experiment, based on relaxation-optimized spectroscopy, yields best results when applied to residues where the methine–methylene group corresponds to a reasonably isolated spin system, as applies for C, F, Y, W, D, N and H residues in proteins, or the C –C groups in nucleic acids. Splittings can be measured under either isotropic or weakly aligned conditions, yielding valuable structural information both through the ³J couplings and the one-, two- and three-bond dipolar interactions. Dipolar couplings for 10 out of 13 sidechains in GB3 are found to be in excellent agreement with its X-ray structure, whereas one residue adopts a different backbone geometry, and two residues are subject to extensive ₁ rotamer averaging. The abundance of dipolar couplings can also yield stereospecific assignments of the non-equivalent methylene protons. For the RNA oligomer, dipolar data yielded stereospecific assignments for six out of the eight C H₂ groups in the loop region of the oligomer, in all cases confirmed by ¹J ^{1} $$" align="middle" border="0"> J , and H resonating downfield of H .Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0175-z.     

α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction

(S(C5'), R(P)) α,β-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.