Biomedical subjects

G Wagner

Publications and source records attributed to G Wagner.

At least 19 recordsLinked to original sources

PubMed1992

Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2.

Metallothioneins are small cysteine-rich proteins capable of binding heavy metal ions such as Zn2+ and Cd2+. They are ubiquitous tissue components in higher organisms, which tentatively have been attributed both unspecific protective functions against toxic metal ions and highly specific roles in fundamental zinc-regulated cellular processes. In this paper a detailed comparison of the NMR solution structure [Schultze, P., Wörgötter, E., Braun, W., Wagner, G., Vasák, M., Kägi, J. H. R. & Wüthrich, K. (1988) J. Mol. Biol. 203, 251-268] and a recent x-ray crystal structure [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xoung, N. H., Furey, W. F., Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] of rat metallothionein-2 shows that the metallothionein structures in crystals and in solution have identical molecular architectures. The structures obtained with both techniques now present a reliable basis for discussions on structure-function correlations in this class of metalloproteins.

Amino Acid Sequence↗

PubMed1992

Suppression by cholinesterase inhibition of a Ca(2+)-independent efflux of [3H]acetylcholine from the neuromuscular junction of the isolated rat diaphragm.

Endplate preparations of the left rat hemidiaphragm were incubated with [3H]choline to label neuronal acetylcholine stores. Elevation of the concentration (13.5-135 mmol/l) of extracellular potassium chloride (KCl) stimulated the release of [3H]acetylcholine in a concentration-dependent manner. KCl (27 mmol/l) still caused a significant efflux of [3H]acetylcholine in a Ca(2+)-free medium. Inhibitors of cholinesterase (physostigmine, diisopropylfluorophosphate) suppressed by 80% this Ca(2+)-independent efflux of [3H]acetylcholine. Vesamicol (10 mumol/l), the blocker of the vesicular acetylcholine carrier, also suppressed the stimulated, Ca(2+)-independent efflux of [3H]acetylcholine. The inhibitory effect of physostigmine was not prevented by muscarine or nicotine receptor antagonists, but the inhibitory effect was lost when the stimulus strength was increased (81 mmol/l KCl). The present experiments showed cholinesterase inhibition to suppress a Ca(2+)-independent efflux of [3H]acetylcholine, probably by interference with a membrane-bound acetylcholine carrier.

Acetylcholine↗

PubMed1992

Changing the inhibitory specificity and function of the proteinase inhibitor eglin c by site-directed mutagenesis: functional and structural investigation.

Amino acids in the serine proteinase inhibitor eglin c important for its inhibitory specificity and activity have been investigated by site-directed mutagenesis. The specificity of eglin c could be changed from elastase to trypsin inhibition by the point mutation Leu45----Arg (L45R) in position P1 [nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Model building studies based on the crystal structure of mutant L45R [Heinz et al. (1991) J. Mol. Biol. 217, 353-371] were used to rationalize this specificity change. Surprisingly, the double mutant L45R/D46S was found to be a substrate of trypsin and various other serine proteinases. Multidimensional NMR studies show that wild-type eglin c and the double mutant have virtually identical conformations. In the double mutant L45R/D46S, however, the N-H bond vector of the scissile peptide bond shows a much higher mobility, indicating that the internal rigidity of the binding loop is significantly weakened due to the loss or destabilization of the internal hydrogen bond of the P1' residue. Mutant T44P was constructed to examine the role of a proline in position P2, which is frequently found in serine proteinase inhibitors [Laskowski and Kato (1980) Annu. Rev. Biochem. 49, 593-626]. The mutant remains a potent elastase inhibitor but no longer inhibits subtilisin, which could be explained by model building. Both Arg51 and Arg53, located in the core of the molecule and participating in the hydrogen bonding network with residues in the binding loop to maintain rigidity around the scissile bond, were individually replaced with the shorter but equally charged amino acid lysine. Both mutants showed a decrease in their inhibitory potential. The crystal structure of mutant R53K revealed the loss of two hydrogen bonds between the core and the binding loop of the inhibitor, which are partially restored by a solvent molecule, leading to a decrease in inhibition of elastase by 2 orders of magnitude.

Amino Acid Sequence↗

PubMed1992

Mapping of the spectral densities of N-H bond motions in eglin c using heteronuclear relaxation experiments.

A new strategy is used for studying the internal motions of proteins based on measurements of NMR relaxation parameters. The strategy yields values of the so-called spectral density functions J(omega) for N-H bond vectors. The spectral density functions are related to the distribution of frequencies contained in the rotational (overall and internal) motions of these NH bond vectors. No a priori model assumptions about the dynamics are required in this approach. The method involves measurements of six relaxation parameters consisting of 15N longitudinal relaxation rates, transverse relaxation rates of in-phase and antiphase coherence, the relaxation rates of heteronuclear 1H-15N two-spin order, the heteronuclear 1H-15N nuclear Overhauser effects, and longitudinal relaxation rates of the amide protons. The values of the spectral density functions at the five frequencies 0, omega N, omega H + omega N, omega H, and omega H - omega N are determined from the relaxation parameters using analytical relations derived previously [Peng & Wagner (1992) J. Magn. Reson. 98, 308-332]. Here, the method is applied to characterize the backbone dynamics of the 15N-enriched proteinase inhibitor eglin c, a protein of 70 residues. The values for J(0) and J(omega N = 50 MHz) vary significantly with the amino acid sequence, whereas the spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are typically much smaller and show no significant variation with the sequence. The collective behavior of the J(omega) values indicate greater internal motion for the proteinase binding loop residues and the first eight N-terminal residues. The additional internal motion in these regions is in the rate range below 450 MHz. The values of J(omega) are also compared with root mean square deviations (rmsds) of backbone atoms as obtained in NMR structure determinations. Low values of J(0) and J(omega N) are correlated with high rmsds. Spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are small and show no correlation with rmsds. A comparison with the spectral density functions obtained by fitting the experimental data to the functional dependence of the Lipari and Szabo formalism [Lipari & Szabo (1982a) J. Am. Chem. Soc. 104, 4546-4559] is made.

Amino Acid Sequence↗

PubMed1992

Design, synthesis and solution structure of a renin inhibitor. Structural constraints from NOE, and homonuclear and heteronuclear coupling constants combined with distance geometry calculations.

A macrocyclic renin inhibitor was designed using molecular modeling and a model of human renin. The synthesized molecular displayed poor binding affinity. To investigate the reasons for the observed inactivity, the structure of the compound has been studied by NMR spectroscopy and distance geometry. Structural constraints for distance geometry calculations were derived from nuclear Overhauser effects and homonuclear and heteronuclear three bond coupling constants. Homonuclear coupling constants were measured directly from the resolution-enhanced proton spectra and heteronuclear coupling constants were measured from the natural abundance 15N- and 13C-edited TOCSY experiments. One phi angle was determined uniquely by this method and two were reduced to two possible values each. By using a statistical analysis of 400 structures generated with distance geometry, two families of structures were found to be consistent with the NMR data. The solution structures so derived were different from the originally designed structure, including an internal hydrogen bond. This provides a possible explanation for the lack of effectiveness of this compound.

Humans↗

PubMed1992

Solution structure of the DNA-binding domain of Cd2-GAL4 from S. cerevisiae.

The GAL4 protein activates transcription of the genes required for galactose utilization in Saccharomyces cerevisiae. The protein, consisting of 881 amino acids, is dimeric when bound to one of the approximately twofold symmetrical DNA sites present in the galactose upstream activating sequence (UASG). Here we use two-dimensional NMR spectroscopy to determine the structure of an amino-terminal fragment of GAL4 (residues 1-65). This fragment, a monomer in solution, binds as a dimer specifically to UASG-containing DNA. Residues 9-40 form a well defined, compact globular cluster, whereas residues 1-8 and 41-66 show considerable conformational mobility in the absence of DNA. The compact domain contains a motif in which six cysteines, located on two symmetrically related helix/extended strand units connected by a long loop, coordinate two central zinc ions, forming a bimetal-thiolate cluster. The zincs were replaced by NMR-active 113Cd in most of our work and structural parameters are therefore derived from the Cd2-protein. The structure obtained for the GAL4 DNA-binding domain represents a novel DNA-binding motif. Essentially the same conformation is observed for the compact domain in solution using NMR techniques as was seen for the central core of the N-terminal fragment bound to DNA using crystallographic techniques. Thus, the core of the DNA-binding domain changes little upon binding DNA.

Amino Acid Sequence↗

PubMed1992

Sequential 1H NMR assignments of kistrin, a potent platelet aggregation inhibitor and glycoprotein IIb-IIIa antagonist.

Sequence-specific nuclear magnetic resonances assignments have been obtained for the protons of kistrin. Kistrin is a small naturally occurring snake venom protein that inhibits platelet aggregation by blocking the interaction of fibrinogen with the membrane-bound glycoprotein IIb-IIIa (GP IIb-IIIa), a receptor from the integrin family. Kistrin has an Arg-Gly-Asp sequence which is believed to form an adhesion recognition sequence that is essential for activity. Therefore, the interaction between kistrin and GP IIb-IIIa may provide important information on the motif used by integrins to recognize their target proteins which bear RGD sequences. Kistrin consists of 68 residues and contains six intramolecular disulfide bonds. Although one-third of the amide protons are protected from exchange with the solvent, there appears to be little or no regular secondary structure. The large number of NOE's between residues separated by two and three positions in the sequence indicates that the protein contains a large number of tightly packed loops. Along with the sequential assignments, this paper also discusses the construction and use of computerized data bases for manipulating NMR results. A strategy for computer-assisted sequential resonance using these data bases is also presented.

Amino Acid Sequence↗

PubMed1992

Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution.

Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient 1H resonance dispersion. Medium- and long-range NOE's have been used to characterize the secondary structure of the binary ligand-enzyme complex in solution.

Amino Acid Sequence↗

PubMed1992

Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints.

The solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and a temperature of 28 degrees C has been determined from NMR data, using distance geometry calculations and restrained energy minimization. The structure determination is based on 730 conformational constraints derived from NMR data, including 644 NOE-derived upper bound distance constraints, constraints on the ranges of 32 dihedral angles based on measurements of vicinal coupling constants, and 54 upper and lower bound constraints associated with nine hydrogen bonds and the three disulfide bonds. The distance geometry interpretation of the NMR data is based on previously published sequence-specific 1H resonance assignments [Montelione et al. (1988) Biochemistry 27, 2235-2243], supplemented here with individual assignments for some side-chain amide, methylene, and isopropyl methyl protons. The molecular architecture of mEGF is the same as that described previously [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230], but the structure is overall more precisely determined by a more extensive set of NMR constraints. Analysis of proton NMR line widths, amide proton exchange rates, and side-chain 3J(H alpha-H beta) coupling constants provides evidence for internal motion in several regions of the mEGF molecule. Because mEGF is one member of a large family of homologous growth factors and protein domains for which X-ray crystal structures are not yet available, the atomic coordinates resulting from the present structure refinement (which we have deposited in the Brookhaven Protein Data Bank) are important data for understanding the structures of EGF-like proteins and for further detailed comparisons of these structures with mEGF.

Amino Acid Sequence↗

PubMed1992

NMR studies of structure and dynamics of isotope enriched proteins.

Structural studies of globular proteins by nmr can be enhanced by the use of isotope enrichment. We have been working with proteins enriched with 15N, and with both 15N and 13C. Due to the isotope enrichment we could assign several large proteins with up to 186 residues and could address structural questions. Furthermore, we can accurately measure heteronuclear and homonuclear vicinal coupling constants. This involves in part multidimensional multiple resonance experiments. This is important for characterization of minor conformational changes caused by mutations. We have also made use of isotope enrichment to study the internal mobility of proteins. We also have developed novel methods for measuring accurately 15N relaxation parameters, in particular transverse relaxation rates. This has led us toward a method for directly mapping spectral density functions of the rotational motions of N-H bond vectors in proteins. The protein systems that are discussed include the unlabeled proteins kistrin and cytochrome c551, and the labeled proteins eglin c, a flavodoxin, and human dihydrofolate reductase.

Bacterial Proteins↗

PubMed1992

Very early acute myocardial infarct treated with streptokinase and balloon angioplasty.

A 57-year-old man developed anginalike chest pain for the first time but there was no objective evidence of an infarct (i.e., EKG and serum enzymes were normal). After 12 days the pain increased, but EKG and serum enzymes remained normal ("preinfarct," crescendo, unstable, or accelerated angina). At this time a cardiac catheterization showed 90% occlusion of the left anterior descending (LAD) coronary artery. On the 17th day after the onset of pain, severe pain recurred together with an abnormal EKG and the patient was taken immediately to the laboratory where a total occlusion of the LAD was now found and he was treated with intracoronary streptokinase. The artery remained open for only a short time, and balloon angioplasty was performed. However, the patient died 12 hours after onset of the last episode of severe pain. A very early acute myocardial infarct was diagnosed at autopsy together with severe coronary atherosclerosis especially of the LAD which had disruption of atherosclerotic plaques and microscopic evidence of embolization.

Angioplasty, Balloon↗

PubMed1992

The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X-ray structures.

A high-precision solution structure of the elastase inhibitor eglin c was determined by NMR and distance geometry calculations. A large set of 947 nuclear Overhauser (NOE) distance constraints was identified, 417 of which were quantified from two-dimensional NOE spectra at short mixing times. In addition, a large number of homonuclear 1H-1H and heteronuclear 1H-15N vicinal coupling constants were used, and constraints on 42 chi 1 and 38 phi angles were obtained. Structure calculations were carried out using the distance geometry program DG-II. These calculations had a high convergence rate, in that 66 out of 75 calculations converged with maximum residual NOE violations ranging from 0.17 A to 0.47 A. The spread of the structures was characterized with average root mean square deviations ( ) between the structures and a mean structure. To calculate the unbiased toward any single structure, a new procedure was used for structure alignment. A canonical structure was calculated from the mean distances, and all structures were aligned relative to that. Furthermore, an angular order parameter S was defined and used to characterize the spread of structures in torsion angle space. To obtain an accurate estimate of the precision of the structure, the number of calculations was increased until the and the angular order parameters stabilized. This was achieved after approximately 40 calculations. The structure consists of a well-defined core whose backbone deviates from the canonical structure ca. 0.4 A, a disordered N-terminal heptapeptide whose backbone deviates by 0.8-12 A, and a proteinase-binding loop whose backbone deviates up to 3.0 A. Analysis of the angular order parameters and inspection of the structures indicates that a hinge-bending motion of the binding loop may occur in solution. Secondary structures were analyzed by comparison of dihedral angle patterns. The high precision of the structure allows one to identify subtle differences with four crystal structures of eglin c determined in complexes with proteinases.

Amino Acid Sequence↗

PubMed1992

Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA-binding domain as studied by amide proton exchange.

Backbone amide proton exchange rates in the DNA-binding domain of GAL4 have been determined using 1H-15N heteronuclear correlation NMR spectroscopy. Three forms of the protein were studied-the native Zn-containing protein, the Cd-substituted protein, and a Zn-GAL4/DNA complex. Exchange rates in the Zn-containing protein are significantly slower than in the Cd-substituted protein. This shows that Cd-substituted GAL4 is destabilized relative to the native Zn-containing protein. Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA-recognition module are significantly reduced by the presence of DNA. In all forms of the protein, the internal dyad symmetry of the DNA-recognition module of GAL4 is reflected by the backbone amide proton exchange rates.

Amino Acid Sequence↗

PubMed1992

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris.

Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding "vesicles" from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO4, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO4 (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO4 for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).

Calcium↗

PubMed1992

A genetic marker at the glucokinase gene locus for type 2 (non-insulin-dependent) diabetes mellitus in Mauritian Creoles.

The prevalence of Type 2 (non-insulin-dependent) diabetes mellitus is high in Mauritius, a multiethnic island nation in the southwestern Indian Ocean. Evaluation of candidate genes in the different ethnic groups represents a means of assessing the genetic component. As glucokinase is known to be a key regulator of glucose homeostasis in liver and pancreatic Beta-cells, the human gene was isolated and a dinucleotide repeat (CA)n marker was identified at this locus. A polymerase chain reaction assay was developed, and alleles differing in size were observed in individuals, according to the number of repeats in the amplified fragment. Eighty-five Creoles and 63 Indians of known glucose tolerance status were typed by amplification of genomic DNA for this dinucleotide (CA)n repeat marker. Four different alleles were observed including Z, the most common allele, and Z + 2, Z + 4, and Z + 10, which differed from Z by 2, 4, and 10 nucleotides respectively. In Mauritian Creoles, the frequency of the Z + 2 allele was greater in Type 2 diabetic subjects than in control subjects (23.8% vs 8.9%, p = 0.008), and the frequency of the Z allele was lower in Type 2 diabetic subjects (60% vs 75.6%, p = 0.03). Analysis with univariate logistic regression models indicated that the Z + 2 allele had the highest odds ratio, 3.08 (95% confidence interval 1.14-8.35, p = 0.0416), among the other risk factors (age, sex, body mass index, and waist/hip ratio). The multivariate odds ratio for Type 2 diabetes was 2.88 (95% confidence interval 0.98-8.50, p = 0.0551).(ABSTRACT TRUNCATED AT 250 WORDS)

Africa↗

PubMed1992

The Iowa Medical Information Network: a concept.

Iowa Methodist Medical Center (IMMC) was one of the nation's early developers of a "hospital/physician" system, entering the arena during October 1985, under the direction of Ginny Wagner. Their "MOLI-Link" system now serves over 150 physicians in 50 locations, and is currently undergoing significant enhancement. MOLI-Link was originally developed using Annson Systems (IBAX) products, and has recently migrated to Wallace Computer Services (Wal-Link) software. Over the past 4 years, IMMC has investigated wide-area medical information networking, and is now actively investigating development of the Iowa Medical Information Network. It is envisioned that IMIN would provide clinical, administrative, and educational information services throughout the IMMC 37 county referral area.

Hospital Information Systems↗

PubMed1992

Application of a microbial sensor for determination of short-chain fatty acids in raw milk samples.

A microbial sensor system, based on the use of immobilized Arthrobacter nicotiana and an oxygen electrode, was applied to determine free short-chain fatty acids in raw milk samples and the result was compared with gas chromatography (GC) and a titrimetric method. The sensor response was linearly related to the concentration of short-chain fatty acids obtained by GC (n = 10, r = 0.92) and to the total concentration of free fatty acids obtained by titrimetric measurement (n = 10, r = 0.78). This result suggests that the present microbial sensor can selectively determine free short-chain fatty acids in raw milk samples and may be useful as a very fast detection method of rancidity in milk.

Animals↗

PubMed1992

A triple-resonance pulse scheme for selectively correlating amide 1HN and 15N nuclei with the 1H alpha proton of the preceding residue.

A 3D 1H-15N-13C triple resonance experiment is presented that contains exclusively cross peaks between the 1HN and 15N nuclei of one residue with the H alpha of the preceding residue. The pulse sequence, designed to minimize the time coherence, is transverse on nuclei with short T2 values. The experiment consists of coherence transfers via one-bond couplings from the HN via N, CO, C alpha to the H alpha and back to the HN for detection; it is called HN(COCA)HA. The experiment was tested on uniformly 15N- and 13C-enriched T4 lysozyme.

Amides↗