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Biomedical subjects

M E Johnson

Publications and source records attributed to M E Johnson.

At least 19 recordsLinked to original sources
PubMed1992

Bivalent-metal binding to CheY protein. Effect on protein conformation.

CheY is a 14 kDa cytoplasmic protein that is activated by the transfer of a phosphoryl moiety to Asp-57 from phosphoCheA during signal transduction in bacterial chemotaxis. It has been established that metal ions are necessary for the autophosphorylation of CheA, the transfer of phosphate from phosphoCheA to CheY and the autodephosphorylation of phosphoCheY. In this work, paramagnetic relaxation enhancement has been used in conjunction with one- and two-dimensional n.m.r. to study the interaction of CheY with bivalent metal ions. These studies have led to the discovery of two conformations of the protein in water, corresponding to the metal-free and the metal-bound states. Binding of bivalent cations like Mg2+, Ca2+, Sr2+, Zn2+ and Mn2+ results in a conformational change from the metal-free to the metal-bound state. Preliminary assignments of the aromatic proton resonances are reported. Comparison of phase-sensitive double-quantum-filtered COSY, homonuclear Hartmann-Hahn coherence transfer and nuclear Overhauser enhancement spectra from the metal-bound and metal-free protein indicates that Trp-58, Thr-87 and Tyr-106 are particularly affected by the conformational change involved, and that this change is limited to a small number of residues. In addition, homonuclear Hartmann-Hahn coherence transfer experiments with paramagnetic Mn2+ show significant suppression of cross-peaks associated with Trp-58 and several neighbouring residues. Comparison of the distances estimated using n.m.r. with the CheY crystal structure indicates that the n.m.r. results are consistent with bivalent metal binding at the cluster of aspartic acid residues that includes Asp-13 and Asp-57. These studies also demonstrate the utility of paramagnetic metal-induced relaxation in conjunction with two-dimensional n.m.r. measurements for exploring ligand-binding sites.

Amino Acid Sequence↗
PubMed1992

Specificity and affinity of binding of phosphate-containing compounds to CheY protein.

1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.

Adenine↗
PubMed1992

Design, synthesis and conformational analysis of gamma-turn peptide mimetics of bradykinin.

Gamma-turns are regular secondary structure elements, found with some frequency in small peptides, that have been implicated in the biologically active conformations of several systems. This report describes the design, synthesis and conformational analysis of a non-peptide gamma-turn mimetic. Low energy conformations of the mimetic system exhibit good conformational agreement with an experimentally observed peptide gamma-turn. The mimetics were incorporated into the nonapeptide bradykinin, for which a gamma-turn, formed by residues Ser 6 to Phe 8, has been hypothesized to be a bioactive conformation. The results indicate that a bioactive conformation of bradykinin may include a reverse turn at this position.

Amino Acid Sequence↗
PubMed1992

Design and synthesis of a CD4 beta-turn mimetic that inhibits human immunodeficiency virus envelope glycoprotein gp120 binding and infection of human lymphocytes.

Poor bioavailability, rapid degradation, antigenicity, and high cost often limit the use of proteinaceous pharmaceuticals. One goal of structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high-resolution structural analysis and rapid modification. The dissection of complex proteins into small synthetic conformationally restricted components is an important step in the design of low molecular weight nonpeptides that mimic the activity of the native protein. We have developed a reverse-turn mimetic system to explore peptide and protein structure-function relationships. We now report the design and synthesis of a small molecule (M(r) 810, as its trifluoroacetate salt), water soluble, proteolytically stable mimetic of residues Gln40-Thr45 of the complementarity-determining 2-like region of CD4. This mimetic has a low micromolar Kd for human T-lymphotropic virus type IIIB gp120 and reduces syncytium formation.

Binding, Competitive↗
PubMed1992

Peptide mimetics of the thrombin-bound structure of fibrinopeptide A.

Recent work has suggested that the thrombin-bound conformation of fibrinopeptide A exhibits a strand-turn-strand motif, with a beta-turn centered at residues Glu-11 and Gly-12. Our molecular modeling analysis indicates that the published fibrinopeptide conformation cannot bind reasonably to thrombin but that reorientation of two residues by alignment with bovine pancreatic trypsin inhibitor provides a good fit within the deep thrombin cleft and satisfies all of the experimental nuclear Overhauser effect data. Based on this analysis, we have successfully designed and synthesized hybrid peptide mimetic substrates and inhibitors that mimic the proposed beta-turn structure. The results indicate that the turn conformation is an important aspect of thrombin specificity and that our turn mimetic design successfully mimics the thrombin-bound conformation of fibrinopeptide.

Amino Acid Sequence↗
PubMed1992

Location of potential binding sites on deoxy hemoglobin for the design of antigelling agents.

The binding sites of indole-based gelation inhibitors with sickle cell hemoglobin were investigated by two parallel theoretical approaches. A geometric approach originated by Kuntz and co-workers uses a spatial buildup scheme to locate potential binding regions, while a hybrid grid/geometric search method searches for specific indole ring binding pockets over the hemoglobin surface. The binding sites derived from these calculations were tested for their ability to accommodate indole rings by means of accessibility calculations with probes of various radii. These sites were further scanned for van der Waals' overlap and electrostatic interactions. A full 5BrTrp residue was built in each indole ring binding site, and its conformational energy of association with sickle hemoglobin was calculated at that site. Our theoretical results predict a total of 14 potential binding regions, including all of the sites observed from X-ray crystallography, and sites that are consistent with solution nuclear magnetic resonance studies.

Antisickling Agents↗
PubMed1992

Diagnostic use of emergency department echocardiogram in massive pulmonary emboli.

Massive pulmonary embolism represents a small percentage of patients presenting to the emergency department with clinical symptoms suggestive of embolic phenomenon. Definitive therapy with thrombolytics or embolectomy is usually delayed until angiographic verification of the clinical suspicion can be obtained. A case is described in which embolectomy was based on clinical diagnosis and confirmatory echocardiogram obtained in the ED. The rapid diagnostic role of echocardiographic data in a subset of patients presenting with clinical symptoms suggestive of massive pulmonary emboli deserves further evaluation.

Acute Disease↗
PubMed1992

Survival of Listeria monocytogenes during the manufacture and ripening of Swiss cheese.

Rindless Swiss cheese was made from a mixture of pasteurized whole and skim milk that was inoculated to contain 10(4) to 10(5) cfu of Listeria monocytogenes (strain Ohio, California, or V7)/ml. During clotting of milk, numbers of L. monocytogenes remained nearly unchanged. When the curd was heated gradually to attain the cooking temperature (50 degrees C), numbers of L. monocytogenes increased by approximately 40 to 45% over those in inoculated milk. Cooking curd at 50 degrees C (122 degrees F) for 30 to 40 min resulted in resilient curd having a pH of 6.40 to 6.45 and decreased L. monocytogenes by 48% compared with numbers of the pathogen in inoculated milk. After curd was pressed under whey, numbers of L. monocytogenes increased by approximately 52% over those in inoculated milk and reached their maxima at the end of this stage. A sharp decrease in numbers of L. monocytogenes occurred during brining of cheese blocks (7 degrees C for 30 h). The population of L. monocytogenes continued to decrease during cheese ripening. Average D values for strains California, Ohio, and V7 were 29.2, 24, and 22.5 d, respectively. Listeria was not detected (direct plating, and cold enrichment) after 80, 77, and 66 d of ripening of Swiss cheese made from milk inoculated with strains California, Ohio, and V7, respectively. Thus, Swiss cheese made in this study did not permit extended survival of L. monocytogenes.

Animals↗
PubMed1992

The oocyte nucleus isolated in oil retains in vivo structure and functions.

We describe a technique for isolating the nucleus of the giant amphibian oocyte under paraffin oil. The method precludes the losses of small solutes and proteins that accompany isolation of nuclei into aqueous media. An individual oocyte is blotted, placed under oil, punctured near the animal pole and then squeezed to gently extrude the nucleus into the oil, thereby avoiding exposure to any aqueous environment. Light and electron microscopy of the oil-isolated nucleus demonstrate that its in vivo morphology is preserved. We also describe techniques that facilitate the study of nuclear functions under oil. Oil-isolated oocyte nuclei retain many in vivo functions for several hours, including size-selective envelope permeability, RNA synthesis and the ability to break down in response to cdc2/cyclin meiotic maturation promoting factor.

Animals↗
PubMed1979

Spin-label techniques for monitoring macromolecular rotational motion: empirical calibration under nonideal conditions.

Practical techniques are demonstrated for determining rotational correlation times of macromolecules from the first harmonic absorption electron spin reasonance spectra of tightly bound spin labels. The techniques are developed to compensate for such nonideal conditions as residual label motion, temperature dependence of rigid limit spectral parameters, and the presence of inhomogeneous line broadening. These effects are all shown to be of importance in monitoring the rotational motion of carbonmonoxyhemoglobin which is spin labeled with the tightly bound nitroxide label, 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy. Spin-label interactions with other paramagnetic agents are also shown to produce spectral changes which are qualitatively similar to, but quantitatively different from, those resulting from increases in the rate of rotational motion.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Tertiary structure variability within the quaternary states of hemoglobin: a spin label study.

Using variable temperature techniques, the spin label spectral resolution of hemoglobin labeled at the beta93 cysteines with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodonacetamide has been greatly enhanced. The effects of different ligands, inositol hexaphosphate, pH and salt concentration upon spin labeled ferrous and ferric hemoglobin indicate that the beta chain tertiary structure exhibits considerable variability within the oxy and deoxy quaternary structures. From these studies ligand and spin state changes both appear to be of significance in producing structural changes; binding of inositol hexaphosphate then produces further structural changes secondary in amplitude.

Chemical Phenomena↗
PubMed1978

Librational motion of an "immobilized" spin label: hemoglobin spin labeled by a maleimide derivative.

The spin label Tempo-maleimide, when "immobilized" in hemoglobin, is shown to exhibit motional fluctuation whose amplitude and/or frequency depend on temperature and solution conditions. These motional fluctuations are observable by several electron spin resonance techniques. For desalted hemoglobin the fluctuations are detectable at approximately -15 degrees C using saturation transfer techniques and at approximately +25 degrees C using line-width measurements of normal absorption spectra. In ammonium sulfate precipitated hemoglobin, however, motional fluctuations are not detectable by either technique up to at least 40 degrees C. The most probable mechanism for spin-label motion appears to be either fluctuations in protein conformation which affect the label binding site or conformational transitions of the nitroxide ring itself. These motional fluctuations are shown to introduce a librational character to the overall label motion during hemoglobin rotational diffusion, with the librational motion significantly affecting the use of spin-label spectral shapes to calculate hemoglobin rotational correlation times.

Chemical Precipitation↗
PubMed1978

Spin label detection of intermolecular interactions in carbonmonoxy sickle hemoglobin.

With recently developed spin label techniques for monitoring macromolecular rotational motion, heme-liganded sickle cell hemoglobin in the presence of inositol hexaphosphate is shown to exhibit restricted motional freedom as compared to liganded normal adult human hemoglobin. This motional restriction is dependent on both hemoglobin concentration and temperature.

Adult↗
PubMed1977

Persistence of antibiotics in milk from cows treated late in the dry period.

Nineteen cows were treated via intramammary infusion at various times prior to calving with one of two dry cow antibiotic preparations (cloxacillin or penicillin). Milk samples from these cows after calving were analyzed for antibiotic residues by the Sarcina lutea cylinder plate method. Eight cows were treated with cloxacillin was detected in the milk of only two cows after calving and then for only one milking, so no problem should exist with cloxacillin if the manufacturer's recommendations are followed. Penicillin residues were not detected in milk samples taken from cows treated more than 9 days prior to calving with the dry cow penicillin preparation. However, seven of nine cows treated with penicillin up to 9 days prior to calving had detectable penicillin residues in the milk from treated quarters after calving. Only one cow had detectable penicillin residues longer than the manufacturer's recommended milk withholding time of 96 h, and this cow was treated only 3 days prior to parturition. Normal routine on a dairy farm and adherence to manufacturer's recommendations should result in no antibiotic residue problems in milk from cows treated during the dry period for mastitis control with either cloxacillin or penicillin.

Animals↗