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M L Hackert

Publications and source records attributed to M L Hackert.

At least 19 recordsLinked to original sources
PubMed1992

Structure determination and refinement of homotetrameric hemoglobin from Urechis caupo at 2.5 A resolution.

A 5 A resolution multiple isomorphous replacement solution for hemoglobin isolated from Urechis caupo revealed a previously unobserved quaternary structure for tetrameric hemoglobin [Kolatkar, Meador, Stanfield & Hackert (1988). J. Biol. Chem. 263(7), 3462-3465]. We report here the structure of Urechis hemoglobin in the cyanomet state refined to 2.5 A resolution by simulated annealing yielding R = 0.148 for reflections F greater than 3 sigma between 5.0 and 2.5 A resolution. The starting model was fitted to a map originally derived from multiple-wavelength anomalous-dispersion phases to 3 A resolution that was then subjected to cyclic twofold molecular averaging and solvent flattening. Structural analysis of the resultant model shows that the unique quaternary assemblage is possible due to several favorable interactions between subunits, including salt links, hydrophobic pockets and interactions mediated by bound water. The tetramer is stabilized by subunit-subunit interactions between the G/H turns and D helices within the crystallographic dimer, and the A/B turn regions and E helices between subunits related by a molecular twofold axis. Interestingly, each subunit has one cysteine residue (Cys21) located in the A/B turn. These twofold-related cysteinyl residues are near enough to one another to form a disulfide bridge but do not.

Animals↗
PubMed1991

Dipotassium and sodium/potassium crystalline picrate complexes with the crown ether 6,7,9,10,12,13,20,21,23,24,26,27-dodecahydrodibenzo[b,n]-[1,4, 7,10,13,16,19,22]octaoxacyclotetracosin (dibenzo-24-crown-8).

The crystal structures of the dipotassium and the mixed sodium/potassium picrate complexes with the crown ether dibenzo-24-crown-8 (DB24C8) were solved and found to be nearly identical. (I): NaK-pic2(DB24C8), [NaK(C6H2N3O7)2(C24H32O8)]. Mr = 966.8, triclinic, P1, a = 8.164 (2), b = 9.960 (2), c = 13.368 (3) A, alpha = 103.92 (3), beta = 108.03 (2), gamma = 93.23 (2) degrees, V = 993.0 (7) A3, Z = 1, Dm = 1.54 (T = 298 K). Dx = 1.62 (1) g cm-3, lambda = (Mo K alpha) 0.71069 A, mu = 2.37 cm-1, F(000) = 500, T = 103 K, R = 0.086 for 2904 unique reflections. (II): K2pic2(DB24C8), [K2(C6H2N3O7)2(C24H32O8)]. Mr = 982.9, triclinic, P1, a = 8.231 (4), b = 9.850 (2), c = 13.346 (4) A, alpha = 103.91 (2), beta = 106.82 (3), gamma = 93.37 (2) degrees, V = 995.7 (9) A3, Z = 1, Dm = 1.59 (T = 298 K), Dx = 1.638 (8) g cm-3, lambda (Mo K alpha) = 0.71069 A, mu = 3.30 cm-1, F(000) = 508, T = 163 K, R = 0.042 for 4835 unique reflections. Both structures feature eight-coordinated cations between alternating layers of relatively flat crown ligands and paired picrates. In the mixed-metal system the two cations are disordered between two P1-related sites; these metal sites have a coordination environment only slightly different from that in the dipotassium structure. Na+ is able to occupy an environment similar to that of K+ under the conditions of these crystals, a situation not previously observed in the chemistry of crown ethers or macrocylic multidentates.

Chelating Agents↗
PubMed1991

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: the utility of methylamine tungstate stain and Butvar support film in the study of macromolecules by transmission electron microscopy.

The structure of ornithine decarboxylase (Mr approximately 1.04 x 10(6] from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted +/- 45 degrees. The two edge views were related by a 30 degrees rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-A resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10 degrees with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.

Image Processing, Computer-Assisted↗
PubMed1989

Branched-chain alpha-keto acid dehydrogenase complex from bovine kidney: radial distribution of mass determined from dark-field electron micrographs.

Scanning transmission electron microscopy (STEM) was used to determine the radial distribution of mass within the bovine kidney branched-chain alpha-keto acid dehydrogenase complex (E1-E2) and its core enzyme, dihydrolipoamide acyltransferase (E2). The particle mass of E2 measured by STEM is (1.19 +/- 0.02) x 10(6). Assuming 24 subunits per E2 core, this value corresponds to a subunit molecular weight of (4.96 +/- 0.08) x 10(4), which agrees well with the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 5.2 x 10(4) (Pettit et al., 1978) and that deduced from the gene sequence, 46,518 (Griffin et al., 1988). Thus, the STEM data reaffirms the 24-subunit model for this E2. Previous studies indicated that the E2 subunits contain an extended, outer lipoyl-bearing domain connected by a trypsin-sensitive segment to a compact, inner catalytic domain. The assemblage of 24 inner domains comprises a cubelike inner core. The quantity and spatial distribution of mass determined from STEM images for the E2 inner core are consistent with this model. The lipoyl-bearing domains are shown to occupy a zone defined by radii of 80-130 A over which the lipoyl moiety may range. This zone overlaps the positions of the 24 branched-chain alpha-keto acid dehydrogenase (E1) molecules, which apparently are located on the of the cubelike inner core.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1989

Pyruvoyl-dependent histidine decarboxylase. Active site structure and mechanistic analysis.

The structure of the pyruvoyl-dependent histidine decarboxylase has been refined to 2.5 A resolution by the methods of x-ray crystallography from crystals grown at pH 4.8, where the enzyme is optimally active. Models of the active site with and without the bound substrate analog, histidine methyl ester (HisOMe), or the product, histamine, have been produced. Comparison of native and ligand-bound structures reveals no widespread differences in conformation but does reveal motion of a few key residues (Tyr-62', Ile-59', Ser-81) upon binding of HisOMe in the active site. The HisOMe binds with the appropriate alpha-carbon-carbon bond oriented as required to facilitate the formation of the transition state. The binding site contains two pockets, one for the imidazole group, and another for the -COOMe group. In the imidazole pocket, the imidazolium group forms hydrogen bonds with two neighboring carboxylates, Asp-63' and the carboxyl terminus of the beta chain, Ser-81. Hydrophobic contacts are also observed. The carboxylate pocket is predominantly hydrophobic as predicted by Alston and Abeles (Alston, T. A., and Abeles, R. H. (1987) Biochemistry 26,4082-4085), but includes one carboxyl group, that of Glu-197, about 3.5 A from the substrate carboxylate. If Glu-197 is protonated under these conditions, it could serve as the proton donor following decarboxylation; if it is ionized under these conditions, its carboxylate group is appropriately placed to enhance the lability of the substrate carboxylate ion by providing a "push" in promoting the flow of electrons that results in decarboxylation. These and other structural features of the binding complex are discussed as they relate to a proposed mechanism of decarboxylation.

Amino Acid Sequence↗
PubMed1989

Crystallization and molecular symmetry of ornithine decarboxylase from Lactobacillus 30a.

Ornithine decarboxylase from Lactobacillus 30a is representative of the large subunit (80 kDa), oligomeric, pyridoxal phosphate-dependent amino-acid decarboxylases. Yellow crystals of ornithine decarboxylase are obtained from polyethylene glycol solutions and belong to space group P6 with unit cell constants a = b = 194.9 and c = 97.44 A, alpha = beta = 90 degrees and gamma = 120 degrees, V = 3.21 x 10(6) A3. Still photographs show reflections at better than 2.4-A resolution. Electron micrographs reported by Guirard and Snell (Guirard, B.M., and Snell, E.E. (1980) J. Biol. Chem. 255, 5960-5964) reveal that the ornithine decarboxylase dodecamer is a hexagonally shaped particle with a point-to-point distance of approximately 210 A and a thickness of approximately 70 A. The crystallographic unit cell can thus accommodate one 10(6)-Da dodecamer (Vm = 3.2 A3/Da), implying that a dimer occupies an asymmetric unit. Tanaka rotation function analysis, using native data (5-7 A) collected from three crystals, reveals that the particle has the expected 622 molecular symmetry with molecular 2-fold axes lying at 20 degrees and 50 degrees from a in the a-b plane. A search for suitable heavy atom derivatives is underway.

Crystallization↗
PubMed1988

Novel subunit structure observed for noncooperative hemoglobin from Urechis caupo.

Tetrameric hemoglobin from the "fat innkeeper" worm Urechis caupo possesses a novel subunit arrangement having an "inside out" quaternary structure in that the G/H helices are located on the outer surface of the tetramer. A 5-A resolution crystal structure reveals that although the individual subunits are beta-like, having a distinct D helix and the general myoglobin fold, the subunit contacts are very different from those previously observed for hemoglobins. Furthermore, the hemoglobin from U. caupo is also quite different from the unusual hemoglobin tetramer from clam which also has its G/H helices on the outer surface but with the hemes in close proximity through E-F helical contacts (Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316, 277-280).

Animals↗
PubMed1986

Crystal structure analysis and refinement at 2.5 A of hexameric C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum. The molecular model and its implications for light-harvesting.

The crystal structure of the light-harvesting protein-pigment complex C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum has been determined by Patterson search techniques on the basis of the molecular model of C-phycocyanin from Mastigocladus laminosus. The crystal unit cell (space group P321) contains three (alpha beta)6 hexamers centred on the crystallographic triads. The hexamer at the origin of the unit cell exhibits crystallographic 32 point symmetry. The other two hexamers (independent of the former) show crystallographic 3-fold and local 2-fold symmetry. The 3-fold redundancy of the asymmetric unit of the crystal cell was used in the refinement process, which proceeded by cyclic averaging, model building and energy-restrained crystallographic refinement. Refinement was terminated with a conventional crystallographic R-value of 0.20 with data to 2.5 A resolution. The two independent hexamers of the unit cell are identical within the limits of error at all levels of aggregation. Two trimers, which closely resemble the M. laminosus C-phycocyanin, are aggregated head-to-head to form the hexamer. Both trimers fit complementarily and are held together by polar and ionic interactions. Conservation of the amino acid residues involved in protein-chromophore and intermonomer interactions suggests common structural features for all biliproteins. Most probably, the hexameric aggregation form present in the crystals is closely related to the discs of native phycobilisome rods. All tetrapyrrole chromophores are extended but with different geometries enforced by different protein surroundings. In particular, interactions of the propionic side-chains with arginine residues and of the pyrrole nitrogen atoms with aspartate residues define configuration and conformation of the chromophores. Relative chromophore distances and orientations have been determined and a preferential pathway for the energy transfer suggested. Accordingly, within a hexamer the absorbed energy is funneled to chromophore B84 and then transduced via B84 chromophores along the phycobilisome rods.

Amino Acid Sequence↗
PubMed1985

Structure determination of histidine decarboxylase from Lactobacillus 30a at 3.0 A resolution.

The crystal structure of histidine decarboxylase from Lactobacillus 30a has been determined by X-ray diffraction methods to a resolution of 3.0 A. This protein is a pyruvoyl-dependent enzyme that is formed by an unusual self-activation process. The structure was determined from an electron density map calculated using multiple isomorphous replacement phases from two heavy-atom derivatives and included contributions from anomalous scattering measurements. The final mean figure of merit was 0.79, based on 28,805 independent reflections. The molecule has an (alpha beta)6 subunit composition and crystallizes in the space group 14122 with a = b = 221.7 A and c = 107.1 A. There is one (alpha beta)3 half molecule per asymmetric unit. The (alpha beta)6 particle is dumbbell-shaped, with each (alpha beta)3 unit being approximately spherical, with a diameter of about 65 A. There is a large central cavity approximately 30 A deep around the molecular 3-fold axis of the (alpha beta)3 unit. The 3-fold related active site pockets are located around the bottom of this cavity and are separated from each other by a distance of approximately 23 A. The inner portion of each (alpha beta) unit, which lies near the interface between the two (alpha beta)3 particles, consists mainly of random coil with several small helical and sheet regions. The outer region of each (alpha beta) unit has an unusual structure consisting of two overlapping, predominantly antiparallel beta-pleated sheets, lined on each side by an alpha-helix. The walls of the central cavity are formed by the 3-fold repeat of two strands from this beta-sandwich structure and one of the helices.

Carboxy-Lyases↗
PubMed1979

Preliminary crystallographic data on monomeric and dimeric hemoglobins from the sea cucumber, Molpadia arenicola.

Large single crystals of two distinct globin chains from coelomic cells of the sea cucumber Molpadia arenicola have been prepared and examined by x-ray crystallography. These hemoglobins exhibit a variety of ligand-dependent association states with the met-hemoglobins existing as monomers and liganded hemoglobins as dimers at physiological concentrations. Monomeric methemoglobin C chain crystallizes in space group P21, with a = 46.0 A, b = 45.3 A, c = 40.9 A, beta = 104.5 degrees, and one monomer per asymmetric unit. These crystals exhibit unusual spectroscopic behavior when examined with a polarizer, turning colorless in certain orientations. This implies that all the heme rings are nearly parallel within the crystals. Dimeric cyanmethemoglobin D chain crystallizes in space group P41212 (P43212), with a = b = 77.0 A, c = 61.5 A, and one-half a dimer per asymmetric unit. These homodimers thus possess a molecular 2-fold which is aligned with the crystallographic dyad.

Animals↗
PubMed1977

Purification and crystallization of NADP+-specific isocitrate dehydrogenase from Escherichia coli using polyethylene glycol.

A simple and rapid method is presented for purifying the NADP+-dependent isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), from Escherichia coli, which relies on fractionation of the enzyme with polyethylene glycol. The shortened preparation results in a 32% relative recovery of purified enzyme at a specific activity of 127 micronmol/min per mg of protein. The Km values for threo-DS-isocitrate, NADP+, NAD+, Mg2+ and Mn2+ are 6.4, 36, 3000, 19.7 and 2.0 micronM, respectively. The stability of the enzyme as a function of dilution and temperature are also reported. Recrystallization of the purified enzyme under different conditions readily produces a variety of single crystals. Crystals grown from ammonium sulfate solutions belong to monoclinic space group C2 with a = 125 A, b = 111 A, c = 83.5 A and beta = 108degrees 45'. Density measurements of these crystals indicate there are two 80 000-dalton dimers per asymmetric unit.

Crystallization↗