Solution structures of polcalcin Phl p 7 in three ligation states: Apo-, hemi-Mg2+-bound, and fully Ca2+-bound.

Polcalcins are small EF-hand proteins believed to assist in regulating pollen-tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain-swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg(2+), or 100 µM Ca(2+). Phl p 7 is monomeric in all three ligation states. In the apo-form, both EF-hand motifs reside in the closed conformation, with roughly antiparallel N- and C-terminal helical segments. In 5.0 mM Mg(2+), the divalent ion is bound by EF-hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca(2+)-bound Phl p 7 resembles that previously reported for Bet v 4-likewise exposing apolar surface to the solvent. Occluded in the apo- and Mg(2+)-bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF-hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at -x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca(2+). When Mg(2+) resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer-sphere ligand. (15)N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states.

Investigating structural alterations in pyrogallol[4]arene-pyrene nanotubular frameworks.

Small-angle neutron scattering (SANS) and diffusion NMR studies are performed to investigate the stability and geometry of hydrogen-bonded pyrene-guest-containing C-hexylpyrogallol[4]arene (PgC(6) -pyrene) nanotubular frameworks in solution. In the solid state, hydrogen-bonded pyrogallol[4]arene tubes are formed; however, the scattering data for PgC(6) -pyrene assemblies in acetone are best modeled as dimeric spheres of PgC(6) with no pyrene guest. The result of diffusion NMR study also indicates the rearrangement of tubular entity into spherical framework in acetone. This is the first example of structural transformation of pyrogallol[4]arene nanotubes (guest-exo) in solution. Individual hydrogen-bonded spheres of PgC(6) exhibits a uniform radius of ca. 8.6 Å and a diffusion coefficient of 9.12 × 10(-10) m(2) s(-1) in acetone. The diffusion NMR measurements further gave, for the first time, insights into how the type of solvent (acetone vs. methanol vs. acetontitrile/D(2) O) governs the structural differences in these nanoassemblies. Solution-phase structural alteration observed for PgC(6) -pyrene gives evidence of enhanced stability of pyrogallol[4]arene nanocapsules over nanotubes.

Solution structure of zinc-seamed C-alkylpyrogallol[4]arene dimeric nanocapsules.

The structural stability and solution geometry of zinc-seamed-C-propylpyrogallol[4]arene dimers has been studied in solution using in situ neutron scattering and 2D-DOSY NMR methods. In comparison with the structures of the analogous copper-/nickel-seamed dimeric entities, the spherical geometry of the PgC3Zn species (R = 9.4 Å; diffusion coefficient = 1.05 × 10-10 m2 s-1) is larger due to the presence of ligands at the periphery in solution. This enhanced radius in solution due to ligation is also consistent with the findings of model molecular dynamics simulations of the zinc-seamed dimers.

Conversion of Pregabalin to 4-Isobutylpyrrolidone-2.

Solid-state studies of C-butyl-resorcin[4]arene with pregabalin (Lyrica, Nervalin) in nitrobenzene yielded a cocrystal of C-butyl-resorcin[4]arene with 4-isobutylpyrrolidone-2. A combined experimental and quantum chemical investigation was implemented to further our understanding of the factors affecting the conversion process.

15N nuclear magnetic resonance relaxation studies on rat beta-parvalbumin and the pentacarboxylate variants, S55D and G98D.

15N relaxation data for Ca(2+)-bound rat beta-parvalbumin (a.k.a. oncomodulin) were analyzed using the Lipari-Szabo formalism and compared with existing data for rat alpha-parvalbumin. Although the average S(2) values for the two proteins are very similar (0.85 for alpha, 0.84 for beta), residue-by-residue inspection reveals systematic differences. alpha tends to have the lower S(2) value in helical regions; beta tends to have the lower value in the loop regions. Rat beta was also examined in the Ca(2+)-free state. The 59 assigned residues displayed an average order parameter (0.90) significantly greater than the corresponding residues in the Ca(2+)-loaded form. The pentacarboxylate variants of rat beta-S55D and G98D-also were examined in the Ca(2+)-bound state. Although both mutations significantly heighten Ca(2+) affinity, they utilize distinct energetic strategies. S55D improves the Ca(2+)-binding enthalpy; G98D improves the binding entropy. They also show disparate peptide backbone dynamics. Whereas beta G98D displays an average order parameter (0.87) slightly greater than that of the wild-type protein, beta S55D displays an average order parameter (0.82) slightly lower than wild-type beta. Furthermore, whereas just two backbone N-H bonds in beta G98D show internal motion on the 20-200-psec timescale, fully 52 of the 93 residues analyzed in beta S55D show this behavior. These findings suggest that the increased electrostatic repulsion attendant to introduction of an additional carboxylate into the CD site ligand array impedes backbone vibrational motion throughout the molecule.

Isolation and identification of an allelopathic phenylethylamine in rice.

Allelopathy is the process whereby an organic chemical (allelochemical) released from one plant influences the growth and development of other plants. Allelochemicals produced by specific rice (Oryza sativa L.) cultivars have potential to manage barnyard grass (Echinochloa crus-galli L.), a major yield-limiting weed species in rice production systems in Asia and North America. In this study, isolation and identification of an allelopathic compound, N-trans-cinnamoyltyramine (NTCT), in a Vietnamese rice cultivar 'OM 5930' was accomplished through bioassay-guided purification using reversed-phase liquid chromatography coupled with spectroscopic techniques, including tandem mass spectrometry, high resolution mass spectrometry, as well as one-dimensional and two-dimensional (1)H NMR and (13)C NMR spectroscopy. The identified compound, NTCT is considered a ß-phenylethylamine. NTCT inhibited root and hypocotyl growth of cress (Lepidium sativum L.), barnyard grass and red sprangletop (Leptochloa chinensis L. Nees) at concentrations as low as 0.24 µM. The ED50 (concentration required for 50% inhibition) of NTCT on barnyard grass root and hypocotyl elongation were 1.35 and 1.85 µM, respectively. Results further demonstrated that mortality of barnyard grass and red sprangletop seedlings was >80% at a concentration of 2.4 µM of NTCT. By 20 days after transplanting, 0.425 nmol of NTCT per OM 5930 rice seedling was released into the culture solution. With concentrations of 42 µg g(-1) fresh weight, production of NTCT in intact rice plants can be considered high. These findings suggest that developing plants of Vietnamese rice cultivar OM 5930 release NTCT and may be utilized to suppress barnyard grass in rice fields. The potency of NTCT may encourage development of this compound as a bio-herbicide.