A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Four New C19-Diterpenoid Alkaloids from Aconitum nagarum Stapf

Four new aconitine-type C₁₉-diterpenoid alkaloids, were isolated from the roots of Aconitum nagarum Stapf which were named as nagarutines A–D ( 1–4 ), together with eleven known compounds ( 5–15 ). The structures of the compounds were identified by IR, HR-ESI-MS, 1D and 2D NMR spectra. All compounds were tested for the inhibitory effect on LPS induced NO production in RAW 264.7 macrophages, compound 7 showed moderate anti-inflammatory activity effect and Inhibition rate is about 44.50%.     

THE FINE STRUCTURE OF TWO PHOTOSYNTHETIC SPECIES OF DINOPHYSIS (DINOPHYSIALES,DINOPHYCEAE)1

Two closely related, photosynthetic species belonging to the genus Dinophysis were examined, D. acuminata Claparède et Lachmann and D. fortii Pavillard. Typical dinoflagellate features include the amphiesmal covering enclosing the cells and the structure of the nucleus and mitochondria. Many other characteristics seem to be specific to the order Dinophysiales. Many rhabdosomes are present, and complex mucocysts are found beneath the amphiesma. The thecal pores are unusual with the base of the pore occluded by a thin disc that is continuous with the main amphiesmal plate. The structure of the apical pore is also distinctive. Chloroplasts are grouped together in chromatospheres, enclosed by a double membrane, and contain paired thylakoids with electron dense contents in the lumen. The two pusules are extensive, each branching off the flagellar canal, and consisting of a large antechamber and a number of convoluted sacs. The entrance of each antechamber, and site of an emerging flagellum, is surrounded by a striated fibrous collar. Near the flagellar pore is a prominent microtubular/microbody complex which penetrates deep into the cell cytoplasm. Consideration is given to taxonomic position of the Dinophysiales and also to the nature and origins of the chloroplasts.     

Genetic metabolic polymorphisms and the risk of cancer: a review of the literature

The purpose of this paper is to systematically analyse the design and results of epidemiological studies on the association between various types of cancer (lung, bladder, breast, colon, stomach) and four genetically-based metabolic polymorphisms, involved in the metabolism of several carcinogens (glutathione-S-transferase M1, debrisoquine hydroxylase, N acetyltransferase, aryl hydrocarbon hydroxylase). These inherited polymorphisms usually cause modifications in the quality or quantity of the relevant enzymes. Such enzymes are involved in the activation/inactivation of known carcinogens and seem to modify the extent to which carcinogens interact with DNA in target tissues. Two enzymes, debrisoquine hydroxylase and aryl hydrocarbon hydroxylase, activate procarcinogens to carcinogens (phase I enzymes). The other two, glutathione-S-transferase M1 and N-acetyltransferase, mainly detoxity carcinogenic substances (phase II enzymes). Because of their role as host factors (modulating the action of carcinogens), it has been hypothesized that subjects presenting a specific phenotype for such polymorphisms could be at a greater risk of developing various types of cancer. A number of epidemiological studies have investigated such associations, often with discordant results. We examine and discuss the design of the studies, and present a meta-analysis of the available data.     

The Role of the Cytoskeleton and Dictyosome Activity in the Pulsatory Growth of Nicotiana tabacum and Petunia hybrida Pollen Tubes

Pollen tubes of Nicotiana tabacum and Petunia hybrida show pulsatory growth. Phases of slow growth lasting minutes are interrupted by pulse-like elongations lasting 10–20 seconds involving an increase of growth rate by up to 24-fold. Inhibition of dictyosome activity with brefeldin A or monensin did not result in an inhibition of pulsatory growth but eventually stopped pollen tube elongation. In contrast to this the inhibition of the cytoskeletal elements with cytochalasin D and colchicine caused the pollen tubes to abandon the pulse-like elongations. It was concluded that the activity of the dictyosomes does not have a controlling function in the mechanism of pulsatory growth, even though it is necessary for pollen tube elongation, since cell wall material is provided by secretory vesicles deriving from the Golgi apparatus. In contrast the cytoskeletal elements, actin and microtubules, seem to play an important regulatory role in the pulse-like elongations. In addition, it was observed that during the experiments several pollen tubes burst upon the completion of a pulse-like expansion, indicating on the one hand that the internal turgor is the driving force of the pulse-like expansions. On the other hand, the bursting shows that the pollen tube cell wall is rather weak at the end of a pulse, indicating that at this point of time it is either thinner or less stable than during the slow growth phase or at the beginning of a pulse.     

Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: Complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin

Summary The ¹H, ¹⁵N and ¹³C backbone and ¹H and ¹³C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, ³J_Hn_H coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel -sheet and five -helices. One of the outer regions of the -sheet shows no regular extended conformation, whereas the outer strand 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five -helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the -helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn¹¹, Ser⁶⁸ and Asn⁷² are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.Abbreviations SQ semiquinone - FMN riboflavin 5-monophosphate; nif, nitrogen fixation - TSP 3-(trimethylsilyl)propionate sodium salt - DSS 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt Supplementary Material is available on request, comprising a Materials and Methods section for the expression and purification of the A. chroococcum flavodoxin, a Table S1 containing the parameters of the titration of A. chroococcum flavodoxin with the Fe protein, and a Table S2 containing the ¹⁵N, H^N, ¹³C, ¹H, ¹³C, ¹H and ¹³CO chemical shifts.To whom correspondence should be addressed.     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.