The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain

Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles, Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88, Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE(1-22) indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations, This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.

Characterization of the sequential non-covalent and covalent interactions of the antitumour antibiotic hedamycin with double stranded DNA by NMR spectroscopy

Hedamycin, a member of the pluramycin class of antitumour antibiotics, consists of a planar anthrapyrantrione chromophore to which is attached two aminosugar rings at one end and a bisepoxide-containing sidechain at the other end, Binding to double-stranded DNA is known to involve both reversible and non-reversible modes of interaction. As a part of studies directed towards elucidating the structural basis for the observed 5'-pyGT-3' sequence selectivity of hedamycin, we conducted one-dimensional NMR titration experiments at low temperature using the hexadeoxyribonucleotide duplexes d(CACGTG)(2) and d(CGTACG)(2). Spectral changes which occurred during these titrations are consistent with hedamycin initially forming a reversible complex in slow exchange on the NMR timescale and binding through intercalation of the chromophore. Monitoring of this reversible complex over a period of hours revealed a second type of spectral change which corresponds with formation of a non-reversible complex. Copyright (C) 1999 John Wiley & Sons, Ltd.

Synthesis and complexes of the sexidentate macrocycle 15-methyl-1,4,7,10,13-pentaazacyclohexadecan-15-amine

The potentially sexidentate polyamine macrocycle 15-methyl-1,4,7,10,13-pentaazacyclohexadecan-15-amine (1) was prepared via a copper(II)-templated route from 3,6,9-triazaundecan-1,ll-diamine, formaldehyde and nitroethane which first formed the copper(II) complex of the macrocycle 15-methyl-15-nitro-1,4,7,10,13-pentaazacyclohexadecane (2), reduced subsequently with zinc and aqueous acid to yield 1. The hexaamine 1, with five secondary amine groups in the macrocyclic ring and one pendant primary amine group, forms inert sexidentate octahedral complexes with cobalt(III), chromium(III) and iron(III). An X-ray structure of [Co(1)](ClO4)(3) defines the distorted octahedron of the complex cation and shows it is a symmetrical isomer with all nitrogens bound and the central aza group trans to the pendant primary amine group. The [M(1)](3+) ions are all stable indefinitely in aqueous solution and exhibit spectra consistent with MN6 d(3) (Cr), low-spin d(5) (Fe) and low-spin d(6) (Co) electronic ground states. For each complex, a reversible M(III/II) redox couple is observed. (C) 2000 Elsevier Science S.A. All rights reserved.

Ni-II and Zn-II complexes of the hexadentate macrocyclic ligand cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetra- decane-6,13-diamine

The title pendent-arm macrocyclic hexaamine ligand binds stereospecifically in a hexadentate manner, and we report here its isomorphous Ni-II and Zn-II complexes (both as perchlorate salts), namely (cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine-kappa(6)N)nickel(II) diperchlorate, [Ni(C12H30N6)](ClO4)(2), and (cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine-kappa(6)N)zinc(II) diperchlorate, [Zn(C-12 H30N6)](ClO4)(2). Distortion of the N-M-N valence angles from their ideal octahedral values becomes more pronounced with increasing metal-ion size and the present results are compared with other structures of this ligand.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Structure revision and assignment of absolute stereochemistry of a marine C-21 bisfuranoterpene

The C-21 bisfuranoterpene (-)-isotetradehydrofurospongin-1 (6), previously isolated from a Western Australian Spongia sp., has been reisolated from a specimen of Spirastrella papilosa collected during scientific trawling operations in the Great Australian Eight. A 2D NMR analysis of 6 has prompted reassignment of the published structure 5, while degradation and chiral HPLC analysis have allowed determination of the absolute stereochemistry.

Determination of the stability constants of mercury(II) complexes of mixed donor macrobicyclic encapsulating ligands

The reactions of mercury(II) with the mixed donor encapsulating ligands 3,6,16-trithia-6,11,19-triazabicyclo[6.6.6]icosane (AMN(3)S(3)sar) and 1-amino-8-methyl-6,19-dithia-3,10,13,16-tetraazabicyclo[6.6.6]icosane (AMN(4)S(2)sar) have been studied. NMR ligand-ligand competition experiments with the ligands 1,4,8,11-tetraazaeyclotetradecane ([14]aneN(4)), 1-thia-4,7,10-triazacyclododecane ([12]aneN(3)S) and ethylenediaminetetraacetic acid (EDTA) with AMN(3)S(3)sar and Hg(II) indicated that [14]aneN(4) would be an appropriate competing ligand for the, determination of the Hg(II) stability constant. Calculations indicated the ratio of concentrations of AMN3S3sar, [14]aneN(4) and Hg(II) required for the determination of the stability constant ranged from 1:1:1 to 1:5:1. Refinement of the titration curves yielded log(10)K[Hg(AMN(3)S(3)sar)](2+) = 17.7. A similar competition titration resulted in the determination of the stability constant for the AMN(4)S(2)sar system as log(10)K[Hg(AMN(4)S(2)sar)](2+) = 19.5. The observed binding constants for the mixed N/S donor systems and the hexaaza analogues sar (3,6,10,13,16,19-hexaazabicyclo [6.6.6]icosane) and diamsar (1,8-diamino-3,6,10,13,16,19 -hexazabicyclo [6.6.6] icosane (log(10)K-[Hg(diamsar)](2+) = 26.4; log(10)K[Hg(sar)](2+) = 28.1) differ by approximately ten orders of magnitude. The difference is ascribed not to a cryptate effect but to a mismatch in the Hg-N and Hg-S bond lengths in the N/S systems.

Modelling of post-irradiation events in polymer gel dosimeters

The nuclear magnetic resonance (NMR) spin-spin relaxation time (T-2) is related to the radiation-dependent concentration of polymer formed in polymer gel dosimeters manufactured from monomers in an aqueous gelatin matrix. Changes in T-2 with time post-irradiation have been reported in the literature but their nature is not fully understood. We investigated those changes with time after irradiation using FT-Raman spectroscopy and the precise determination of T-2 at high magnetic field in a polymer gel dosimeter, A model of fast exchange of magnetization taking into account ongoing gelation and strengthening of the gelatin matrix as well as the polymerization of the monomers with time is presented. Published data on the changes of T-2 in gelatin gels as a function of post-manufacture time are used and fitted closely by the model presented. The same set of parameters characterizing the variations of T-2 in gelatin gels and the increasing concentration of polymer determined from Fr-Raman spectroscopy are used successfully in the modelling of irradiated polymer gel dosimeters. Minimal variations in T-2 in an irradiated PAG dosimeter are observed after 13 h.

NMR shim coil design utilising a rapid spherical harmonic calculation method

A rapid spherical harmonic calculation method is used for the design of Nuclear Magnetic Resonance shim coils. The aim is to design each shim such that it generates a field described purely by a single spherical harmonic. By applying simulated annealing techniques, coil arrangements are produced through the optimal positioning of current-carrying circular arc conductors of rectangular cross-section. This involves minimizing the undesirable harmonies in relation to a target harmonic. The design method is flexible enough to be applied for the production of coil arrangements that generate fields consisting significantly of either zonal or tesseral harmonics. Results are presented for several coil designs which generate tesseral harmonics of degree one.

Conformations of platypus venom C-type natriuretic peptide in aqueous solution and sodium dodecyl sulfate micelles

Nuclear magnetic resonance spectroscopy was used to investigate the conformations of the platypus venom C-type natriuretic peptide A (OvCNPa) in aqueous solutions and in solutions containing sodium dodecyl sulfate (SDS) micelles. The chemically synthesized OvCNPa showed a substantial decrease in flexibility in aqueous solution at 10 degreesC, allowing the observation of medium- and long-range nuclear Overhauser enhancement (NOE) connectivities. Three-dimensional structures calculated using these data showed flexible and reasonably well-defined regions, the locations of which were similar in the two solvents. In aqueous solution, the linear part that spans residues 3-14 was basically an extended conformation while the cyclic portion, defined by residues 23-39, contained a series of beta-turns. The overall shape of the cyclic portion was similar to that observed for an atrial natriuretic peptide (ANP) variant in aqueous solution. OvCNPa adopted a different conformation in SDS micelles wherein the N-terminal region, defined by residues 2-10, was more compact, characterised by turns and a helix, while the cyclic region had turns and an overall shape that was fundamentally different from those structures observed in aqueous solution. The hydrophobic cluster, situated at the centre of the ring of the structure in aqueous solution, was absent in the structure in the presence of SDS micelles. Thus, OvCNPa interacts with SDS micelles and can possibly form ion-channels in cell membranes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Conductivity, NMR and crystallographic study of N,N,N,N-tetramethylammonium dicyanamide plastic crystal phases: an archetypal ambient temperature plastic electrolyte material

N,N,N,N-Tetramethylammonium dicyanamide (Me(4)NDCA) has been examined via differential scanning calorimetry (DSC), thermogravimetric analysis, conductivity, single crystal X-ray diffraction and H-1 nuclear magnetic resonance (NMR) analyses, and was found to be highly conductive in the solid state (sigma = 10(-3) S cm(-2) at 420 K) and to also exhibit unusual plastic crystal behaviour. To investigate the correlation between such behaviour and the occurrence of molecular rotations in the crystal, H-1 NMR second moment measurements are compared with calculated values predicted from the crystal structure. While DSC analysis indicates a number of solid-solid transitions at ambient temperatures, subsequent H-1 NMR analysis of the Me4N+ cation shows that a variety of rotational motions become active at low (

Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

in Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage gimel-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2 mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5 mM sample in 10 mM phosphate, pH 6.05, 0.1 M NaCl, recorded at 36 degreesC, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a = b 142.2 Angstrom, c = 192.1 Angstrom, and diffracted beyond 2.7 Angstrom resolution with synchrotron radiation. (C) 2003 Elsevier Inc. All rights reserved.

Visualization of drip channels in meat using NMR microimaging

The progressive changes in the water distribution within rabbit muscles were studied by nuclear magnetic resonance microscopy during the first 24 h postmortem. T-2 images revealed development of interspersed lines with higher signal intensities in the muscle, reflecting formation of channels containing mobile water. The appearance of the interspersed lines progressed throughout the measuring period and became increasingly evident. After about 3 h postmortem the signal intensity also increased in areas near the surface of the samples, which reflects migration of the mobile water to the sample surface. Proton density images showed the presence of a chemical shift artifact in the interspersed lines, implying that the intrinsic development of water channels progressed in close proximity to the connective tissue. (C) 2004 Elsevier Ltd. All rights reserved.

Solution structure of χ-conopeptide MrIA, a modulator of the human norepinephrine transporter

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.: Schroder, C. L; Kaye, D. M.; Adams, D. I.; Alewood, P. F.; Lewis, R. J. J Biol Client 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hypl2 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency. This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by entailing the Biopolymers editorial office at biopolymers@wiley.com (c) 2005 Wiley Periodicals, Inc.