Biomedical subjects
Publications and source records attributed to Geoffrey B Jameson.
Silver(I) complexes of the two closely related linear bis-bidentate pyridazine-based Schiff-base ligands 5 and 6 exhibit very different solid state molecular architectures, easily deformed grid vs. side-by-side, due to steric and electronic factors.
Bovine beta-lactoglobulin (BLG) is a globular protein of uncertain physiological function and a member of the lipocalin superfamily of proteins. Here, we present the X-ray structure at 3.0 angstroms of BLG (variant A) from an orthorhombic (P2(1)2(1)2(1)) pseudo-tetragonal crystal form that suffers from pseudo-merohedral twinning (final R(working) = 0.224, R(free) = 0.265). Crystals were grown by dialysis against ultra-purified water (i.e., at very low ionic strength), at pH approximately 5.2 (approximately pI), conditions vastly different from all other BLG structures determined previously. This allows critical assessment of the BLG structure and of the influence that pH, ionic strength, and crystal packing may have on the molecular structure of BLG. The pH-sensitive EF loop is found in the closed conformation characteristic of BLG at pH less than 7 and moderate to high ionic strength. Although the hydrophobic pocket appears to be empty, the presence of highly disordered water molecules cannot be excluded. The dimer interface and the hydrophobic pocket (calyx) are preserved. However, the orientation of the subunits in the dimer varies considerably with crystal form. Structure is deposited with PDB ID 2akq.
The multimodal ligand hexakis(2-pyridyloxy)cyclotriphosphazene (L) and its 4-methyl-2-pyridyloxy analogue (MeL) react with Ag(I) to afford {[AgL]+}infinity supramolecular cationic columns via self-assembly, with the anions occupying the intercolumnar channels. In contrast, the reaction of MeL with Cu(I) yields a dimetallic Cu(II) complex containing mu-OH and mu-4-methyl-2-pyridyloxylato bridges.
In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-beta-semialdehyde [(S)-ASA] and pyruvate, a reaction catalyzed by dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52). It has been proposed that Arg138, a residue situated at the entrance to the active site of DHDPS, is responsible for binding the carboxyl of (S)-ASA and may additionally be involved in the mechanism of (S)-lysine inhibition. This study tests these assertions by mutation of Arg138 to both histidine and alanine. Following purification, DHDPS-R138H and DHDPS-R138A each showed severely compromised activity (approximately 0.1% that of the wild type), and the apparent Michaelis-Menten constant for (S)-ASA in each mutant, calculated using a pseudo-single substrate analysis, was significantly higher than that of the wild type. This provides good evidence that Arg138 is indeed essential for catalysis and plays a key role in substrate binding. To test whether structural changes could account for the change in kinetic behavior, the solution structure was probed via far-UV circular dichroism, confirming that the mutations at position 138 did not modify secondary structure. The crystal structures of both mutant enzymes were determined, confirming the presence of the mutations and suggesting that Arg138 plays an important role in catalysis: the stabilization of the catalytic triad residues, a motif we have previously demonstrated to be essential for activity. In addition, the role of Arg138 in (S)-lysine inhibition was examined. Both mutant enzymes showed the same IC(50) values as the wild type but different partial inhibition patterns, from which it is concluded that arginine 138 is not essential for (S)-lysine inhibition.
3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and the four-carbon monosaccharide D-erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus is a member of the DAH7PS Ibeta subfamily, which also includes the KDO8PS enzymes. KDO8PS (3-deoxy-D-manno-octulosonate-8-phosphate synthase) catalyzes a closely related reaction of PEP with the five-carbon monosaccharide D-arabinose 5-phosphate (A5P). DAH7PS from P. furiosus requires a metal ion for activity and, unlike other characterized DAH7PS enzymes, is not inhibited by aromatic amino acids. Purified P. furiosus DAH7PS is able to utilize not only the four-carbon phosphorylated monosaccharides E4P and 2-deoxy-D-erythrose 4-phosphate but also the five-carbon phosphorylated monosaccharides A5P, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate with similar kcat but much increased KM values. DL-glyceraldehyde 3-phosphate and D-glucose 6-phosphate are not substrates. The structure of recombinant P. furiosus DAH7PS in complex with PEP was determined to 2.25 A resolution. The asymmetric unit consists of a dimer of (beta/alpha)8-barrel subunits. Analysis of the buried surfaces formed by dimerization and tetramerization, as observed in the crystal structure, provides insight into both the oligomeric status in solution and the substrate ambiguity of P. furiosus DAH7PS. P. furiosus DAH7PS is both the first archaeal and the first "naked" DAH7PS (without N-terminal extensions) to be fully characterized functionally and structurally. The broad substrate specificity of this DAH7PS, the lack of allosteric inhibition, and various structural features indicate that, of the enzymes characterized to date, P. furiosus DAH7PS may be the contemporary protein closest to the ancestral type I enzyme.
Dihydrodipicolinate synthase (DHDPS) mediates the key first reaction common to the biosynthesis of (S)-lysine and meso-diaminopimelate. The activity of DHDPS is allosterically regulated by the feedback inhibitor (S)-lysine. The crystal structure of DHDPS from Escherichia coli has previously been published, but to only a resolution of 2.5 A, and the structure of the lysine-bound adduct was known to only 2.94 A resolution. Here, the crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from E. coli are presented to 1.9 and 2.0 A, respectively, resolutions that allow, in particular, more accurate definition of the protein structure. The general architecture of the active site is found to be consistent with previously determined structures, but with some important differences. Arg138, which is situated at the entrance of the active site and is thought to be involved in substrate binding, has an altered conformation and is connected via a water molecule to Tyr133 of the active-site catalytic triad. This suggests a hitherto unknown function for Arg138 in the DHDPS mechanism. Additionally, a re-evaluation of the dimer-dimer interface reveals a more extensive network of interactions than first thought. Of particular interest is the higher resolution structure of DHDPS with (S)-lysine bound at the allosteric site, which is remote to the active site, although connected to it by a chain of conserved water molecules. (S)-Lysine has a slightly altered conformation from that originally determined and does not appear to alter the DHDPS structure as others have reported.
Racemic 2-deoxyerythrose 4-phosphate was synthesized and one enantiomer of this compound was found to be a substrate for Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. When the reaction was carried out in deuterium oxide, an enzyme-catalyzed regio- and stereoselective incorporation of deuterium into the product was observed.
The tribenzylphosphine (PBz3) complexes of mercury(II), [Hg(PBz3)2](BF4)2, [Hg(PBz3)2(NO3)2] and [HgX(NO3)(PBz3)](X = Cl, Br, I and SCN), have been synthesised and their structures determined by single-crystal X-ray crystallography. [Hg(PBz3)2](BF4)2 contains [Hg(PBz3)2]2+ cations with linear P-Hg-P coordination, the first example of a truly two-coordinate [Hg(PR3)2]2+ complex. The mercury coordination in [Hg(PBz3)2(NO3)2] can be described as distorted tetrahedral, with a significant deviation of the P-Hg-P angle from linearity as a result of coordination of the nitrate groups. Nitrate coordination is also observed in [HgX(NO3)(PBz3)](X = Cl, Br, I), resulting in significantly non-linear P-Hg-X coordination. The thiocyanate complex is a centrosymmetric thiocyanate-bridged dimer with distorted trigonal-pyramidal mercury coordination to the P atom of PBz3, to the S and N atoms of two bridging thiocyanate groups, and to the O atom of one nitrate group. For all the nitrato complexes, secondary mercury-nitrate interactions (Hg-O 2.7-3.1 A) effectively raise the coordination number of the Hg(II) centres to six. High-resolution 31P solid-state NMR spectra of the six tribenzylphosphine mercury(II)-complexes, obtained by combining magic-angle spinning, proton dipolar decoupling and proton-phosphorus cross-polarization (CP-MAS), have been recorded. The spectra of [Hg(PBz3)2](BF4)2 and [HgX(NO3)(PBz3)](X = Cl, Br, I and SCN) exhibit a single line, due to species that contain non-magnetic isotopes of mercury, and satellite lines, due to 1J(31P-199Hg) coupling. The asymmetric unit of [Hg(PBz3)2(NO3)2] contains two molecules with four phosphorus environments, resulting in two AB spectra with 2J(31P-31P) coupling, due to species that contain non-magnetic isotopes of mercury, and satellite lines consisting of two ABX spectra, due to 1J(31P-199Hg) coupling. These spectra have been analysed to yield all of the chemical shifts and coupling constants involved. A remarkable increase in 1J(31P-199Hg) is observed from [Hg(PBz3)2](BF4)2 to [Hg(PBz3)2(NO3)2] as a consequence of the incorporation of the nitrate group into the Hg coordination sphere in the latter case. Several of the spectra also exhibit broader satellites due to the presence of scalar spin-spin coupling between 31P and the quadrupolar 201Hg nucleus. Slow-spinning methods have been used to analyze the spinning-sideband intensities of the NMR spectra, in order to obtain the 31P shielding anisotropy and asymmetry parameters Deltasigma and eta. The 199Hg and 31P NMR shielding tensors of PMe3 models of the above six compounds have been calculated using relativistic density functional theory. The 31P results are in good agreement with experiment and assist in the assignment of some of the signals.
Reactions of tetrahedral Cu(I) and Ag(I) cations with 2,3,4,5-tetra(4-pyridyl)thiophene allows targeted construction of coordination frameworks with zeolite-like, 4(2).8(4), topologies.
In the absence of added base, a deep navy-blue dimeric copper complex [CuII(H2L)(MeCN)]2(BF4)4 (1) of the non-deprotonated bis-terdentate diamide ligand H2L self-assembles whereas in the presence of base a grass-green [2 x 2] grid complex [CuII(HL)]4(BF4)4 (2) of the monodeprotonated ligand HL-, a rare example of a discrete grid of pyrazine-bridged metal ions, is formed.
A new series of meso-substituted diaryl free-base and metalloporphyrins have been prepared. Each arene has been substituted with both a methyl group in the ortho position and a formyl group in the meta position. Rotation of the arene units is prevented at room temperature due to the steric restrictions imposed by the flanking methyl groups at the porphyrin beta-pyrrolic positions on the methyl groups at the ortho position on the meso-substituted arene unit. This allowed the alpha alpha and alpha beta atropisomers of this porphyrin to be separated and characterised. X-Ray crystallographic determination of the structure of the free-base porphyrin revealed a very flat porphyrin core. Metallation resulted in the isolation and characterisation of the nickel, zinc and copper derivatives. The assignments of the alpha alpha and alpha beta isomers are confirmed by X-ray crystallographic determination of the structures of the Cu(II) analogues. The copper alpha alpha structure exhibits a very twisted porphyrin core, the copper alpha beta structure is also distorted, but to a lesser degree. The activation energy for rotation has been calculated for each of the 2H, Ni and Zn derivatives. The energy required to rotate the arene ring increases in the order Ni < Zn approximately 2H. No significant difference in the free energy of rotation was observed between experiments carried out with the alpha alpha and small alpha beta isomers.
The synthesis and characterisation of a pyridazine-containing two-armed grid ligand L2 (prepared from one equivalent of 3,6-diformylpyridazine and two equivalents of p-anisidine) and the resulting transition metal (Zn, Cu, Ni, Co, Fe, Mn) complexes (1-9) are reported. Single-crystal X-ray structure determinations revealed that the copper(I) complex had self-assembled as a [2 x 2] grid, [Cu(I) (4)(L2)(4)][PF(6)](4).(CH(3)CN)(H(2)O)(CH(3)CH(2)OCH(2)CH(3))(0.25) (2.(CH(3)CN)(H(2)O)(CH(3)CH(2)OCH(2)CH(3))(0.25)), whereas the [Zn(2)(L2)(2)(CH(3)CN)(2)(H(2)O)(2)][ClO(4)](4).CH(3)CN (1.CH(3)CN), [Ni(II) (2)(L2)(2)(CH(3)CN)(4)][BF(4)](4).(CH(3)CH(2)OCH(2)CH(3))(0.25) (5 a.(CH(3)CH(2)OCH(2)CH(3))(0.25)) and [Co(II) (2)(L2)(2)(H(2)O)(2)(CH(3)CN)(2)][ClO(4)](4).(H(2)O)(CH(3)CN)(0.5) (6 a.(H(2)O)(CH(3)CN)(0.5)) complexes adopt a side-by-side architecture; iron(II) forms a monometallic cation binding three L2 ligands, [Fe(II)(L2)(3)][Fe(III)Cl(3)OCl(3)Fe(III)].CH(3)CN (7.CH(3)CN). A more soluble salt of the cation of 7, the diamagnetic complex [Fe(II)(L2)(3)][BF(4)](2).2 H(2)O (8), was prepared, as well as two derivatives of 2, [Cu(I) (2)(L2)(2)(NCS)(2)].H(2)O (3) and [Cu(I) (2)(L2)(NCS)(2)] (4). The manganese complex, [Mn(II) (2)(L2)(2)Cl(4)].3 H(2)O (9), was not structurally characterised, but is proposed to adopt a side-by-side architecture. Variable temperature magnetic susceptibility studies yielded small negative J values for the side-by-side complexes: J=-21.6 cm(-1) and g=2.17 for S=1 dinickel(II) complex [Ni(II) (2)(L2)(2)(H(2)O)(4)][BF(4)](4) (5 b) (fraction monomer 0.02); J=-7.6 cm(-1) and g=2.44 for S= 3/2 dicobalt(II) complex [Co(II) (2)(L2)(2)(H(2)O)(4)][ClO(4)](4) (6 b) (fraction monomer 0.02); J=-3.2 cm(-1) and g=1.95 for S= 5/2 dimanganese(II) complex 9 (fraction monomer 0.02). The double salt, mixed valent iron complex 7.H(2)O gave J=-75 cm(-1) and g=1.81 for the S= 5/2 diiron(III) anion (fraction monomer=0.025). These parameters are lower than normal for Fe(III)OFe(III) species because of fitting of superimposed monomer and dimer susceptibilities arising from trace impurities. The iron(II) centre in 7.H(2)O is low spin and hence diamagnetic, a fact confirmed by the preparation and characterisation of the simple diamagnetic iron(II) complex 8. Mössbauer measurements at 77 K confirmed that there are two iron sites in 7.H(2)O, a low-spin iron(II) site and a high-spin diiron(III) site. A full electrochemical investigation was undertaken for complexes 1, 2, 5 b, 6 b and 8 and this showed that multiple redox processes are a feature of all of them.
The mammalian iron-binding proteins lactoferrin (Lf) and transferrin (Tf) bind iron very tightly, but reversibly. Despite homologous structures and essentially identical iron binding sites, Tf begins to release iron at pH 6.0, whereas Lf retains iron to pH approximately 3.5. This difference in iron retention gives the two proteins different biological roles. Two lysine residues, Lys 206 and Lys 296, which form a hydrogen-bonded dilysine pair in human Tf, have been shown to strongly influence iron release from the N-lobe. The equivalent residues in human Lf are Arg 210 and Lys 301, and we have here mutated Arg 210 in the N-lobe half-molecule of human lactoferrin, Lf(N), to probe its role in iron release. The Lf(N) mutants R210G, R210E, and R210L were expressed, purified, and crystallized, and their crystal structures were determined and refined at resolutions of 1.95 A (R210G), 2.2 A (R210E), and 2.0 A (R210L). The overall structures are very similar to that of wild-type Lf(N), but with small differences in domain orientations. In each of the mutants, however, Lys 301 (equivalent to Lys 296 in Tf) changes its conformation to fill the space occupied by Arg 210 Neta2 in wild-type Lf(N), interacting with the two tyrosine ligands Tyr 92 and Tyr 192. By comparison with other Lf and Tf structures, we conclude that Lys 301 (or Lys 296 in Tf) only occupies this site when residue 210 (206 in Tf) is nonpositive (neutral as in R210G and R210L or negative as in R210E). Thus, Lys 206 in the Tf dilysine pair is identified as having a depressed pK(a). Three specific sites are variably occupied by polar groups in the Lf mutants and other Lf and Tf proteins, and when coupled with iron-release data, these give new insights into the factors that most influence iron retention at low pH.
Human lactoferrin is an iron-binding protein with a bilobal structure. Each lobe contains a high-affinity binding site for a single Fe(3+) ion and an associated CO(3)(2-) ion. Although iron binds very tightly, it can be released at low pH, with an accompanying conformational change in which the two domains move apart. The Arg121Asp (R121D) mutant of the N-lobe half-molecule of human lactoferrin was constructed in order to test whether the Asp121 side chain could substitute for the CO(3)(2-) ion at the iron-binding site. The R121D mutant protein was crystallized in its apo form as it lost iron during crystallization. The crystals were also merohedrally twinned, with a twin fraction close to 0.5. Starting from the initial molecular-replacement solution [Breyer et al. (1999), Acta Cryst. D55, 129-138], the structure has been refined at 3.0 A resolution to an R factor of 13.9% (R(free) of 19.9%). Despite the moderate resolution, the high solvent content and non-crystallographic symmetry contributed to electron-density maps of excellent quality. Weakened iron binding by the R121D mutant is explained by occlusion of the anion-binding site by the Asp side chain. The opening of the two domains in the apoR121D structure (a rotation of 54 degrees ) closely matches that of the N-lobe in full-length lactoferrin, showing that the extent of the conformational change depends on properties inherent to the N-lobe. Differences in the C-terminal portion of the N-lobe (residues 321-332) for apoR121D relative to the closed wild-type iron-bound structure point to the importance of this region in stabilizing the open form.
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