Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 451 records · Page 25Linked to original sources

PubMed1993

The 0.25-nm X-ray structure of the Bowman-Birk-type inhibitor from mung bean in ternary complex with porcine trypsin.

The structure of the Bowman-Birk-type inhibitor from mung bean Phaseolus aureus has been determined in ternary complex with porcine trypsin. The complex formed crystals of the trigonal space group P3(1)21 which diffracted to a resolution of 250 pm. Each of the two mung bean protease reactive sites is bound to trypsin according to the standard mechanism for serine proteinase inhibition. The binding loops thereby adopt the canonical conformation for the standard mechanism; however, the sub-van der Waals contact between the active-site serine O gamma (195) and the P1 carbonyl carbon of both loops is significantly smaller (210 pm) than hitherto observed, with continuous electron density connecting the two atoms. The inhibitor is formed by two double-stranded antiparallel beta-sheets, which are connected into a moderately twisted beta-sheet by a network of hydrogen bonds involving main-chain atoms and two water molecules. All contacts with neighbors in the crystal lattice occur between trypsin molecules. This apparently gives rise to an unusual form of disorder where the complexes pack in two orientations Ta:MaMb:Tb and Tb:MbMa:Ta (Ta, Tb = trypsin, Ma = mung bean loop I, Mb = mung bean loop II), such that the asymmetric unit consists of the ternary complex in two orientations, each with half occupancy. This is nearly equivalent to an asymmetric unit which has one trypsin molecule with full occupancy and one mung bean inhibitor with half occupancy and a crystallographic twofold symmetry axis through its center. Because of the approximate twofold symmetry of the inhibitor itself, however, the electron density was interpretable for most of the inhibitor (17 residues at the termini were not resolved) and shows evidence of its double orientation.

Animals↗

PubMed1993

Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin.

Astacin, a 200 residue digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype of the "astacin family", which comprises several membrane-bound mammalian endopeptidases and developmentally implicated regulatory proteins. Large trigonal crystals of astacin were grown, and X-ray reflection data to 1.8 A resolution were collected. The astacin structure has been solved by multiple isomorphous replacement using six heavy-atom derivatives, and refined to a crystallographic R-value of 0.158 applying stringent constraints. All 200 residues are clearly defined by electron density; 181 solvent molecules have been localized. Besides the native structure, the structures of Hg-astacin (with a mercury ion replacing the zinc) and of the apoenzyme were also refined. The astacin molecule exhibits a kidney-like shape. It consists of an amino-terminal and a carboxy-terminal domain, with a deep active-site cleft in between. The zinc ion, located at the bottom of this cleft, is co-ordinated in a novel trigonal-bipyramidal geometry by three histidine residues, a tyrosine and by a water molecule, which is also bound to the carboxylate side-chain of Glu93. The amino-terminal domain of astacin consists mainly of two long alpha-helices, one centrally located and one more peripheral, and of a five-stranded pleated beta-sheet. The amino terminus protrudes into an internal, water-filled cavity of the lower domain and forms a buried salt bridge with Glu103; amino-terminally extended pro-forms of astacin are thus not compatible with this structure. The carboxy-terminal domain of astacin is mainly organized in several turns and irregular structures. Because they share sequence identity of about 35%, the structures of the proteolytic domains of the other "astacin" members must be quite similar to astacin. Only a few very short deletions and insertions quite distant from the active-site distinguish their structures from astacin. The five-stranded beta-sheet and the two helices of the amino-terminal domain of astacin are topologically similar to the structure observed in the archetypal zinc-endopeptidase thermolysin; the rest of the structures are, in contrast, completely unrelated in astacin and thermolysin. The zinc ion, the central alpha-helix and the zinc-liganding residues His92, Glu93 and His96 of astacin are nearly superimposable with the respective groups of thermolysin, namely with the zinc ion, the "active-site helix", and His142TL, Glu143TL and His146TL of the zinc-binding consensus motif His-Glu-Xaa-Xaa-His (where Xaa is any amino acid residue).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin.

The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis. This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin. M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods. The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin. The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar. The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin. The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution. The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein. Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A. The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed. Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a. The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations. In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcaligenes↗

PubMed1993

Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2.

A new assay for the calcium channel activity of annexin V was developed. The calcium-sensitive fluorescence indicator, fura-2, was incorporated into large unilamellar vesicles (LUV). After establishing a calcium gradient across the liposomal membranes, native or mutated annexin V was added. The resulting calcium influx into the LUV detected through the fluorescence changes of fura-2 was used as a qualitative test for the electrophysiological properties of annexin V.

Annexin A5↗

PubMed1993

Advances in metallo-procarboxypeptidases. Emerging details on the inhibition mechanism and on the activation process.

Our knowledge on the structure and functionality of pancreatic carboxypeptidases is rapidly expanding to include that of their zymogen forms. The recent application of fast and mild isolation procedures, together with modern molecular genetic and biochemical-biophysical characterization approaches, has provided a clearer view of the basic structures and functional states in which these zymogens occur, and their evolutionary relationships. The same holds for related metallo-carboxypeptidases, either in the pro or active forms, that have been isolated and characterized in non-digestive fluids and tissues, where they probably play an important role in protein and peptide processing. The determination of the three-dimensional structure of the A and B pancreatic zymogens has revealed the molecular determinants of their inactivity and proteolytic activation. The folding of their 95-residue activation segment in a globular N-terminal domain (74-81 residues) and in a connecting region (20-14 residues), and the specific contacts of these pieces with the substrate binding sites of the enzyme, are important factors in zymogen inhibition. On the other hand, the different length of the alpha-helical connecting region and the stability of its contacts with the enzyme account for the different activation properties of A and B zymogens.

Amino Acid Sequence↗

PubMed1993

Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24).

The crystal structure of the porcine heart catalytic subunit of cAMP-dependent protein kinase in a ternary complex with the MgATP analogue MnAMP-PNP and a pseudosubstrate inhibitor peptide, PKI(5-24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine-rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three-dimensional space. The structure supports an in-line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.

Adenosine Triphosphate↗

PubMed1993

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).

Amino Acid Sequence↗

PubMed1993

Life in hot springs and hydrothermal vents.

Hot springs and hydrothermal systems occurring within volcanic areas are inhabited by hyperthermophilic microorganisms, some of which grow at temperatures up to 110 degrees C. Hyperthermophiles grow anaerobically or aerobically by diverse metabolic types. Within the high temperature ecosystems, primary production is independent from solar energy.

Archaea↗

PubMed1993

Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum.

A novel rod-shaped hyperthermophilic archaeum has been isolated from a boiling marine water hole at Maronti Beach, Ischia, Italy. It grew optimally at 100 degrees C and pH 7.0 by aerobic respiration as well as by dissimilatory nitrate reduction, forming dinitrogen as a final product. Organic and inorganic compounds served as substrates during aerobic and anaerobic respiration. Growth was inhibited by elemental sulfur. The cell wall was composed of a surface layer of hexameric protein complexes arranged on a p6 lattice. The core lipids consisted mainly of glycerol diphytanyl glycerol tetraethers with various degrees of cyclization. The G+C content was 52 mol%. The new isolate resembled members of the genera Thermoproteus and Pyrobaculum by its ability to form characteristic terminal spherical bodies ("golf clubs"). On the basis of its 16S rRNA sequence, the new isolate exhibited a close relationship to the genus Pyrobaculum. It is described as a new species, which we name Pyrobaculum aerophilum (type strain: IM2; DSM 7523).

Aerobiosis↗

PubMed1993

A warm-water perfusion plaque for hyperthermic treatment of intraocular tumors.

This paper describes the design and performance of a plaque aimed at delivering hot-water hyperthermia to intraocular tumors. The plaque transfers heat into the tissues via conduction. Maximum temperature cannot exceed inflow water temperature. The high water flow rate provided by the hyperthermia unit guarantees an excessive power output at a constant temperature level, resulting in a homogeneous temperature distribution in the tissue. Heat distribution over the plaque surface was shown to be uniform. System performance has been studied on enucleated human as well as on living rabbit eyes. Thermal mapping was done in two and one planes, respectively, and the spatial distribution was calculated. Results promise therapeutic temperature levels to a depth exceeding 10 mm.

Animals↗

PubMed1993

Serpin structures.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1993

[Intramedullary nailing of fractures during the growth period with a new implant].

Intramedullary nailing with flexible titan pins is a simple and safe method for the treatment of fractures in children. Avoiding the epiphysis two pins are introduced into the medullary space in a double-crossing way to stabilise diaphysial fractures of bigger bones such as femur, tibia or humerus. Forearm fractures are splinted with a single pin each to radius and ulna. The method is also practicable in proximal fractures of the radius, reduction and stabilisation can be achieved with an intramedullary pin in one step avoiding arthrotomy.

Adolescent↗

PubMed1992

Impact of protein-protein contacts on the conformation of thrombin-bound hirudin studied by comparison with the nuclear magnetic resonance solution structure of hirudin(1-51).

The impact of protein-protein interactions on the conformation of the N-terminal hirudin domain consisting of residues 1 to 51 in the X-ray crystal structure of a hirudin-thrombin complex was investigated through comparisons with the nuclear magnetic resonance solution structure of hirudin(1-51). The close overall similarity observed between these two structures contrasts with the behavior of the C-terminal 17-residue polypeptide segment of hirudin, which is flexibly disordered in solution but exhibits a defined conformation in the complex with thrombin. Localized structural differences in the N-terminal domain include that residues 1 to 3 of hirudin in the crystalline complex form a hydrogen-bonding network with thrombin that is reminiscent of a parallel beta-sheet. Moreover, the backbone conformation of residues 17 to 20 in the complex does not contain the characteristic hydrogen bond observed for the type II' reverse turn in the solution structure, and the side-chains of Ser19 and Val21 have significantly different orientations in the two structures. Most of these structural changes can be related directly to thrombin-hirudin contacts, which may also be an important factor in the mechanism of hirudin action. In this context, it is of special interest that other residues that also make numerous contacts with thrombin, e.g. Thr4, Asp5 and Asn20, have identical conformations in free hirudin and in the complex.

Hirudins↗

PubMed1992

Isolation, crystallization, crystal structure analysis and refinement of B-phycoerythrin from the red alga Porphyridium sordidum at 2.2 A resolution.

The light-harvesting pigment-protein complex B-phycoerythrin from the red alga Porphyridium sordidum has been isolated and crystallized. B-Phycoerythrin consists of three different subunits forming an (alpha beta)6 gamma aggregate. The three-dimensional structure of the (alpha beta)6 hexamer was solved by Patterson search techniques using the molecular model of C-phycocyanin from Fremyella diplosiphon. The asymmetric unit of the crystal cell (space group P3, with a = b = 111.2 A, c = 59.9 A, alpha = beta = 90 degrees, gamma = 120 degrees) contains two (alpha beta) monomers related by a local dyad. Three asymmetric units are arranged around the crystallographic 3-fold axis building an (alpha beta)6 hexamer, as in C-phycocyanin. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional R-factor of the final model was 18.9% with data to 2.2 A resolution. The molecular structures of the alpha and beta-subunits resemble those of C-phycocyanin. Major changes in comparison to phycocyanin are caused by deletion or insertion of segments involved in protein-chromophore interactions. The singly linked phycoerythrobilin chromophores alpha-84, alpha-140a, beta-84 and beta-155 are each covalently bound to a cysteine by ring A. The doubly linked chromophore beta-50/beta-61 is attached at cysteine beta-50 through ring A and at cysteine beta-61 through ring D. B-Phycoerythrin contains additionally a 30 kDa gamma-subunit, which is presumably located in the central cavity of the hexamer. It is disordered, as a consequence of crystal and local symmetry averaging.

Amino Acid Sequence↗

PubMed1992

Escherichia coli dihydrodipicolinate synthase. Identification of the active site and crystallization.

Escherichia coli dihydrodipicolinate synthase (DHDPS) (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis, catalyses the condensation of pyruvate and aspartate beta-semialdehyde (ASA) by a ping-pong mechanism. Pyruvate binds first to the enzyme, forming a Schiff base with the epsilon-amino group of Lys-161, followed by binding of ASA. Km values of 0.57 and 0.55 mM were determined for pyruvate and DL-ASA respectively. 3-Bromopyruvate inhibits DHDPS with a Ki of 1.6 mM. DHDPS is 50% inhibited by 1.0 mM-L-lysine, 1.2 mM-sodium dipicolinate or 4.6 mM-S-2-aminoethyl-L-cysteine. Crystals of DHDPS diffracting to beyond a resolution of 0.24 nm (2.4 A) were obtained under several experimental conditions. Diffraction patterns were compatible with trigonal space groups P3(1)21 or P3(2)21, with unit-cell parameters a = b = 12.26 nm and c = 11.19 nm. The density of the crystals indicates the presence of a dimer of DHDPS subunits per asymmetric unit.

Amino Acid Sequence↗

PubMed1992

The effect of metal binding on the structure of annexin V and implications for membrane binding.

The structure of annexin V, crystallised in the presence of two calcium or barium ions for each protein molecule, was solved by molecular replacement to 0.24 nm resolution. The two metal ions are found in domains I and IV, i.e. on the same side of the channel that lies in the centre of the molecule. The structures of the barium and calcium form are extremely close, the only differences localised in the metal-binding sites that lie on the surface of the molecule. The occupancies of the metal ions, however, are lower for barium than for calcium, expressing the lower affinity of the protein for the former. The packing of the annexin molecules in the crystal asymmetric unit may represent a model for the calcium driven association of membrane-bound annexins that leads to membrane fusion.

Annexin A5↗

PubMed1992

Complete sequential 1H and 15N nuclear magnetic resonance assignments and solution secondary structure of the blue copper protein azurin from Pseudomonas aeruginosa.

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14,000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH 5.5 and 40 degrees C by using homo- and heteronuclear two-dimensional (2D) and three-dimensional (3D) nuclear magnetic resonance spectroscopic experiments. Combined analysis of a 3D homonuclear 1H Hartmann-Hahn nuclear Overhauser (3D 1H HOHAHA-NOESY) spectrum and a 3D heteronuclear 1H nuclear Overhauser 1H[15N] single-quantum coherence (3D 1H[15N] NOESY-HSQC) spectrum proved especially useful. The latter spectrum was recorded without irradiation of the water signal and provided for differential main chain amide (NH) exchange rates. NMR data were used to determine the secondary structure of azurin in solution. Comparison with the secondary structure of azurin obtained from X-ray analysis shows a virtually complete resemblance; the two beta-sheets and a 3(10)-alpha-3(10) helix are preserved at 40 degrees C, and most loops contain well-defined turns. Special findings are the unexpectedly slow exchange of the Asn-47 and Phe-114 NH's and the observation of His-46 and His-117 N epsilon 2H resonances. The implications of these observations for the assignment of azurin resonance Raman spectra, the rigidity of the blue copper site, and the electron transfer mechanism of azurin are discussed.

Amino Acid Sequence↗

PubMed1992

X-ray crystallographic characterisation of type-2-depleted ascorbate oxidase from zucchini.

The type-2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X-ray crystallography and EPR spectroscopy. The X-ray structure analysis by difference-Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR-active type-2 copper is depleted most; type-1 copper is not affected. This observation indicates preferential formation of a 1 Cu-depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu-depleted species may represent an anti-ferromagnetically coupled copper pair which is EPR-silent and could explain the disappearance of the type-2 EPR signal.

Ascorbate Oxidase↗