Identification of white truffle species using RAPD markers

Morphologically very similar species of white truffle were analyzed by the random amplified polymorphic DNA (RAPD) technique. Species-specific RAPD fragments were selected and pairs of primers were designed on their sequences. Sequence-characterized amplified regions were developed and applied to identify these species throughout their entire life cycle: fruit body, mycelium, ectomycorrhiza. This procedure provides an unambiguous and rapid tool for typing species whose morphology is very similar.     

2D 1H NMR studies of oxidized 2(Fe4S4) ferredoxin from Clostridium pasteurianum

Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the beta-CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys beta-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding alpha-CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.     

Incorporation of nitrate nitrogen into amino acids during the anaerobic germination of rice

Summary Incorporation of¹⁵NO₃-into amino acids was studied during the anaerobic germination of rice seeds. In treated coleoptiles, the label was incorporated into glutamine, glutamate, alanine,-aminobutyric acid (Gaba), arginine, aspartate and methionine. These findings are consistent with a primary incorporation of nitrate nitrogen into glutamine, glutamate and aspartate, and their further conversion to alanine, Gaba, arginine and methionine.     

The solution structure of cytochrome c 6 from the green alga Monoraphidium braunii

 The three-dimensional structure in solution of the reduced form of cytochrome c ₆ from the green alga Monoraphidium braunii has been solved through NMR data. Cytochrome c ₆ acts as a small mono-heme electron carrier protein between the two membrane-embedded complexes cytochrome f and photosystem I. The structure was determined using 1278 relevant interproton NOEs out of 1776 assigned NOEs with distance geometry (DG) calculations which included 36 stereospecific assignments and 20 experimentally found angle constraints. The family of structures obtained from the DG calculations was subjected to energy minimization and molecular dynamics simulation using previously defined force field parameters for the heme and its ligands. In all stages of the calculations, the solution structure is well defined and similar to the now available X-ray structure. Received: 18 January 1996 / Accepted: 19 April 1996     

Circular dichroism and 1H NMR studies of Co2+- and Ni2+-substituted concanavalin A and the lentil and pea lectins

Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.     

Notice

¹⁷O NMR studies of ¹⁷O enriched water solutions containing superoxide dismutase have been performed between pH 7.5 and 11.7. Whereas T₁ measurements do not reveal any interaction between ¹⁷O and the paramagnetic copper center, the linewidth results appreciably increased with increasing pH with an apparent pK_a of 11.3. Comparison with ¹H NMR relaxation studies allows to interpret the present data as due to binding to the copper ion of an OH^? anion at high pH. The binding position should be of “equatorial” type, not involving the binding position of the coordinated water.     

NOE and two-dimensional correlated 1H-NMR spectroscopy of cytochrome c' from Chromatium vinosum.

1H two-dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) spectra of cytochrome c' from Chromatium vinosum have been obtained. The protein is of medium size (Mr 28,000), essentially high spin (S = 5/2) although some quantum mechanical spin admixing with S = 3 2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (alpha-CH2 propionate and beta-CH2 protons of the bound histidine) and between alpha-CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c' structures and analogous systems.     

An investigation of Cu2Zn2 superoxide dismutase and its Ile-137 mutant at high pH

The activity profile of the CU₂Zn₂HSOD Ile-137 mutant has a pK_a of 9.6, i. e. one unit lower than the wild type (WT). This property has allowed us to investigate the inactive high pH form of the enzyme before denaturation occurs. The electronic and EPR spectra do not change with the above pK_a. The ¹H NMR spectrum of the CU₂CO₂-analog reveals slight decreases in the hyperfine shifts of the protons of His-48 at high pH, which are consistent with a water molecule becoming closer to the copper ion, as detected through water ¹H T ₁ ^–1 NMR measurements. The affinity of azide at high pH is lower than at low pH, though still sizeable. The WT follows the same pattern up to pH pK_a. It appears that the drop in activity is not related to any major change involving the metal coordination sphere, but is related to changes in the electrostatic potential due to the deprotonation process. Offprint requests to: I. Bertini