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Y K Shin

Publications and source records attributed to Y K Shin.

At least 73 records · Page 4Linked to original sources
PubMed1997

Transient channel-opening in bacteriorhodopsin: an EPR study.

Active translocation of ions across membranes requires alternating access of the ion binding site inside the pump to the two membrane surfaces. Proton translocation by bacteriorhodopsin (bR), the light-driven proton pump in Halobacterium salinarium, involves this kind of a change in the accessibility of the centrally located retinal Schiff base. This key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonation switch, spin labels were attached to pairs of engineered cysteine residues in the cytoplasmic interhelical loops of bR. Light-induced changes in the distance between a spin label on the EF interhelical loop and a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-resolved electron paramagnetic resonance (EPR) spectroscopy. Both distances increase transiently by about 5 A during the photocycle. This opening occurs between proton release and uptake, and may be the conformational switch that changes the accessibility of the retinal Schiff base to the cytoplasmic surface after proton release to the extracellular side.

Bacteriorhodopsins↗
PubMed1997

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling.

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.

ATP-Binding Cassette Transporters↗
PubMed1997

The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR.

Hemagglutinin (HA) is a homotrimeric surface glycoprotein of the influenza virus. In infection, it induces membrane fusion between viral and endosomal membranes at low pH. Each monomer consists of the receptor-binding HA1 domain and the membrane-interacting HA2 domain. It has been known that the NH2-terminal region of the HA2 domain, the so-called "fusion peptide", inserts into the target membrane and plays a crucial role in triggering fusion between the viral and endosomal membranes. A major portion of the HA2 domain (FHA2: aa 1 to 127) of influenza virus X-31, including the NH2-terminal fusion peptide region, was expressed in Escherichia coli. Through site-directed mutagenesis, eight cysteine (Cys) mutants in the fusion peptide region of HA2 (A5C, I6C, A7C, G8C, I10C, N12C, G13C, W14C) were generated and modified with a nitroxide spin label. Using spin-labeling electron paramagnetic resonance (EPR) techniques, we investigated the conformation, membrane topology and the local oligomeric state of the fusion peptide region in the membrane. EPR spectra showed that this region is likely to exist as a flexible monomer in the membrane at both neutral and fusogenic pH conditions. In addition, EPR power saturation methods allowed us to measure the depth in the membrane of the spin label at each mutation site. The resulting depth profile is consistent with an alpha-helix tilted approximately 25 degrees from the horizontal plane of the membrane with a maximum depth of 15 A from the phosphate group. The tilt and rotational orientation correlates well with a calculated amphiphilicity of this region.

Amino Acid Sequence↗
PubMed1997

Breath pentane concentrations during labor and the effect of epidural analgesia on the pentane concentration.

Increased lipid peroxidation has been observed in pregnancy and particularly in preeclampsia. Pentane, a by-product of lipid peroxidation, can be measured in exhaled breath, and its measurement is considered a non-invasive method of assessing lipid peroxidation in vivo. We measured pentane levels in the breath of 36 healthy parturient women and examined the effect of epidural analgesia on the pentane level. Single-expiratory breath samples were analyzed by gas chromatography. The breath pentane level was higher during labor (4.88 parts per billion [p.p.b.], 95% confidence interval 3.25-6.51 p.p.b.) than before the induction of labor (3.10 p.p.b., 95% confidence interval 2.01-4.19 p.p.b.). There was a significant decrease in the pentane level after the institution of epidural analgesia (2.27 p.p.b., 95% confidence interval 1.43-3.11 p.p.b.). Our results suggest that labor may be accompanied by an increase in lipid peroxidation, and epidural analgesia reverses this increase.

Journal Article↗
PubMed1997

Reclassification of Nocardioides simplex ATCC 13260, ATCC 19565, and ATCC 19566 as Rhodococcus erythropolis.

Our phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences and chemotaxonomic analyses showed that Nocardioides simplex ATCC 13260, ATCC 19565, and ATCC 19566 are more closely related to the genus Rhodococcus, especially Rhodococcus erythropolis, than to the genus Nocardioides. N. simplex ATCC 13260 and N. simplex ATCC 19565 and ATCC 19566 exhibited levels of 16S rDNA similarity of 99.4 and 100%, respectively, to R. erythropolis DSM 43066T. Strains ATCC 13260, ATCC 19565, and ATCC 19566 had mesodiaminopimelic acid in their peptidoglycan and MK-8(H2) as their predominant menaquinone. These three strains produced cellular fatty acid patterns similar to those of R. erythropolis strains rather than those of Nocardioides species. Therefore, N. simplex ATCC 13260, ATCC 19565, and ATCC 19566 should be reclassified as strains of R. erythropolis Gray and Thornton 1928.

DNA, Bacterial↗
PubMed1997

Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov.

The taxonomic position of a misclassified strain, Zoogloea remigera IAM 12670T (= ATCC 25925T = P. R. Dugan 115T), was reevaluated. A phylogenetic analysis based on 16S ribosomal rDNA sequences revealed that this organism was located in the beta subclass of the class Proteobacteria with members of the genus Telluria as its closest relatives. On the basis of phenotypic and phylogenetic information, we propose that this organism should be reclassified in a new taxon with the name Duganella zoogloeoides gen. nov., sp. nov.

Culture Media↗
PubMed1997

Retinoblastoma which developed in microphthalmia.

We report a rare case of retinoblastoma which developed in unilateral microphthalmia. A 4-year-old girl presented with a large, intraocular mass in microphthalmia without a family history of retinoblastoma. The eyeball was enucleated due to rapid growth of the intraocular tumor. Microscopically it proved to be a retinoblastoma with some Flexner-Wintersteiner rosettes. This unusual case shows that retinoblastoma should not be excluded just because the eye is small, particularly if the family history is not available or is incomplete.

Child, Preschool↗
PubMed1996

HIV-1 gp41 tertiary structure studied by EPR spectroscopy.

HIV gp41 is the transmembrane glycoprotein responsible for fusion of viral and cellular membranes, enabling viral entry. The structure of gp41 was studied using two synthetic peptides derived from the ectodomain of gp41: a 38-residue peptide from the "heptad repeat" region (hr.wt), and a 34-residue peptide from a region closer to the C-terminus (bt wt). These peptides were found to form a trimer of heterodimers with approximately 80% alpha-helicity. To study their alignment, distances between spin-labels attached to Cys residues on Cys-substituted peptides were measured using a recently-developed electron paramagnetic resonance method [Rabenstein, M.D., & Shin, Y.-K. (1995) Proc Natl. Acad. Sci. U.S.A. 92, 8239-8243]. The heterotrimeric peptides were found to be antiparallel, consistent with a study on proteolytically cleaved peptide fragments of gp41 [Lu, M., Blacklow, S.C., & Kim, P.S. (1995) Nat. Struct. Biol. 2, 1075-1082]. Furthermore, the C-terminal 19 residues of hr.wt are not apposed to bt.wt, and 15 residues of bt.wt extend beyond the end of br.wt. Consistent with this alignment are tertiary interactions between specific sites of these peptides probed by spin-label mobility. Additionally, a second pair of peptides was studied. From the model, these are expected to align with complete overlap. Alone, neither was helical, but when mixed, they were 83% helical. Based on the alignment of the peptides, a model of the prefusogenic form of gp41 was constructed which is significantly different from the structure of influenza hemagglutinin.

Amino Acid Sequence↗
PubMed1996

Temperature dependence of polypeptide partitioning between water and phospholipid bilayers.

Various thermodynamic forces (e.g., the hydrophobic effect, electrostatic interactions, peptide immobilization, peptide conformational changes, "bilayer effects," and van der Waals dispersion forces) can participate in the transfer of polypeptides from aqueous solution into lipid bilayers. To investigate the contributions of these forces to peptide-membrane thermodynamics, we have studied the temperature dependence of the water-bilayer partitioning of 4 polypeptides derived from the first 25 amino acid residues in the presequence of subunit IV of yeast cytochrome c oxidase (Cox IVp) using electron paramagnetic resonance spectroscopy. The partitioning of the Cox IVp peptides into phospholipid bilayers increases as the temperature is increased from 3 to 40 degrees C. The contribution of bilayer surface expansion to the temperature-dependent partitioning is estimated to be relatively small and to contribute minimally to the increased bilayer binding of the peptides with increasing temperature. Thermodynamic analysis of the data shows that the transfer of the peptides from water into bilayers at 298 K is driven by the entropic term (-T delta Str) with values ranging from -6.7 to -10 kcal mol-1, opposed by the enthalpic term (delta Htr) by approximately 4 kcal mol-1, and accompanied by a change in heat capacity (delta Cp) ranging from -117 to -208 cal K-1 mol-1. Our results indicate that while a variety of forces do, in fact, contribute to the transfer free energies (delta Gtr), the major driving force for the water-to-bilayer transfer is the hydrophobic effect.

Amino Acid Sequence↗
PubMed1996

Effects of protein kinase inhibitors on the stimulated neutrophil responses by degraded immunoglobulin G.

Effects of protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride and protein tyrosine kinase inhibitors, genistein, tyrphostin and 2,5-dimethylcinnamate on the neutrophil responses stimulated by immunoglobulin G (IgG), complement C5a or platelet-activating factor were studied. After receptor binding, the role of protein kinase C and protein tyrosine kinase in the stimulation of neutrophil responses, superoxide production and lysosomal enzyme release in degraded IgG-activated neutrophils may be similar to chemoattractant-stimulated cells. In contrast to complement C5a or platelet-activating factor, protein tyrosine kinase appears to play an important role in the regulation of intracellular Ca2+ mobilization in neutrophils activated by degraded IgG rather than by protein kinase C.

Animals↗
PubMed1996

On the dynamics and conformation of the HA2 domain of the influenza virus hemagglutinin.

To investigate the dynamics and conformation of the membrane-interacting HA2 domain of the hemagglutinin protein of influenza virus, the peripheral part of the HA2 domain (aa 1-127) was expressed in Escherichia coli. Four consecutive single-cysteine mutants, F63C, H64C, Q65C, and I66C, were generated using site-directed mutagenesis. This region is proposed to undergo a conformational change from a loop to a helical coiled-coil when going from the native to the fusion-active state [Bullough et al. (1994) Nature (London) 371, 37-43]. In the trimeric coiled-coil geometry positions 63 and 66 belong to the core so that cysteines from individual monomers are spatially close. On the other hand, positions 64 and 65 face the aqueous phase so that cyteines from monomers are spatially remote. The mutants were studied with cysteine-cysteine cross-linking and the spin-labeling electron paramagnetic resonance (EPR) in both the membrane-bound state and in the detergent-solubilized state. Extensive intramolecular cysteine-cysteine cross-linking was observed not only for F63C and I66C but also for H64C. Rates of cross-linking were comparable for these three mutants at physiological temperatures. These results are inconsistent with what is expected for a well-defined coiled-coil and suggest that the region containing the mutation sites is flexible. However, a characteristic cross-linking pattern consistent with a well-defined coiled-coil developed at very low temperatures. Line shapes of EPR spectra also indicate that this region is dynamic at ambient temperatures. Such flexibility perhaps arises from an equilibrium between a coiled-coil and a random coil conformation. No significant changes of the EPR spectra were observed upon lowering the pH to fusogenic conditions, suggesting that this flexible structure is the stable conformation at both neutral and low pH. The dynamic flexibility of this region may have important implications for the mechanism of HA-induced membrane fusion; for example it may be required for the apposition of the viral and endosomal membranes.

Circular Dichroism↗
PubMed1996

Direct determination of the membrane affinities of individual amino acids.

Amino acids have distinct lipid bilayer affinities which influence the insertion and topology of membrane-bound polypeptides and proteins. To measure membrane affinities, 14 uncharged amino acids were introduced individually at a guest site in a 25-residue peptide derived from the membrane-binding presequence of yeast cytochrome c oxidase, and the peptides were labeled with a nitroxide spin-label. The free energies of transfer from phospholipid bilayers to water (delta delta Gbilayer) were determined directly by examination of partitioning into phospholipid bilayers using electron paramagnetic resonance. The delta delta Gbilayer values are in agreement with hydrophobicities assessed from 1-octanol-water partitioning of N-acetyl amino acid amides [Fauchere, J.-L., & Pliska, V. (1983) Eur. J. Med. Chem. 18, 369-375; Eisenberg, D., & McLachlan, A. (1986) Nature 319, 199-203] and quantitatively demonstrate the role of the hydrophobic effect in membrane-protein interactions.

Amino Acid Sequence↗
PubMed1996

The effect of glucagon on spontaneous contractility of isolated pregnant uterine muscle.

OBJECTIVE: To determine whether glucagon has relaxant effects on the spontaneous contractions of term pregnant human uterine smooth muscle in an isolated preparation. METHODS: Myometrial specimens were excised from the upper incisional surface of the lower uterine segment in seven women during elective cesarean delivery. The muscle strips were suspended in tissue baths and isometric tension was recorded. After establishing rhythmic spontaneous contractions, glucagon reconstituted with distilled water or the accompanying diluent was added directly to the bath in a cumulative manner. In the second phase of the study, the effect of the diluent (1.6% glycerin with 0.2% phenol) alone on muscle contractility was evaluated. RESULTS: Glucagon had no effect on uterine muscle concentrations when reconstituted with distilled water. However, glucagon reconstituted with the diluent decreased the contractile amplitude by 27 +/- 11% (mean +/- standard deviation, P < .01) and the frequency by 13 +/- 10% (P < .05) at a concentration of 20 micrograms/mL. At a cumulative concentration of 40 micrograms/mL, the reductions in amplitude and frequency were 65 +/- 13% (P < .001) and 18 +/- 14% (P < .01), respectively. The diluent at equivalent concentrations exerted relaxation similar to that produced by glucagon when reconstituted with the diluent. The relaxant effects were not different between the two solutions (P > .2, power 90%, alpha = .05). CONCLUSION: These results suggest that relaxation of contractions was likely caused by the diluent rather than by glucagon. We conclude that glucagon does not have a direct relaxant effect on spontaneous contraction of isolated uterine muscle obtained from term pregnant uteri.

Adult↗
PubMed1995

A peptide from the heptad repeat of human immunodeficiency virus gp41 shows both membrane binding and coiled-coil formation.

The envelope glycoprotein gp41 from human immunodeficiency virus type 1 (HIV-1) is involved in membrane fusion and virus entry. It contains a functionally important leucine zipper-like heptad repeat region (residues 553-590). To investigate the solution structure and membrane-binding properties of this region, cysteine-substituted variants of a 38-residue peptide derived from the heptad repeat were synthesized and modified with nitroxide spin labels. Analytical equilibrium ultracentrifugation studies indicated it is primarily tetrameric in solution, in contrast to the protein gp160 which is a mixture of trimers and tetramers. Electron paramagnetic resonance (EPR) measurements indicated that the peptide was bound to vesicles containing 10 mol % negatively charged lipids. The peptides were bound parallel to the membrane surface, near the water-membrane interface, in a structure different from the solution structure, most likely as monomers. When Asp, Pro, or Ser was substituted for Ile at the core "a" position of the heptad repeat in the middle of the peptide, the coiled coil was destabilized. In addition, these peptides showed reduced membrane-binding affinities. Thus, mutations that destabilized coiled-coil formation also decreased membrane-binding propensity. These experimental results, taken with previous evidence, suggest two functions for the heptad repeat of gp41 after CD4 binding: (1) to form an extended coiled coil; (2) to provide a hydrophobic face that binds to the host-cell membrane, bringing the viral and cellular membranes closer and facilitating fusion.

Amino Acid Sequence↗
PubMed1995

Determination of the distance between two spin labels attached to a macromolecule.

An EPR "spectroscopic ruler" was developed using a series of alpha-helical polypeptides, each modified with two nitroxide spin labels. The EPR line broadening due to electron-electron dipolar interactions in the frozen state was determined using the Fourier deconvolution method. These dipolar spectra were then used to estimate the distances between the two nitroxides separated by 8-25 A. Results agreed well with a simple alpha-helical model. The standard deviation from the model system was 0.9 A in the range of 8-25 A. This technique is applicable to complex systems such as membrane receptors and channels, which are difficult to access with high-resolution NMR or x-ray crystallography, and is expected to be particularly useful for systems for which optical methods are hampered by the presence of light-interfering membranes or chromophores.

Amino Acid Sequence↗
PubMed1995

A limiting law for the electrostatics of the binding of polypeptides to phospholipid bilayers.

Two cysteine-substituted variants of a peptide derived from the first 25 residues of the presequence for subunit IV of cytochrome c oxidase were synthesized and modified with a nitroxide spin label. The equilibrium partitioning of these spin-labeled peptides into negatively charged phospholipid vesicles was studied with electron paramagnetic resonance (EPR) to investigate the binding energetics. It is found that the binding equilibrium constant is an explicit function of a unique variable, the membrane surface potential psi in the Gouy-Chapman-Stern theory. Moreover, at low psi (< 0.5RT/F) the binding equilibrium is described by the linear dependence of the transfer free energy delta G(el) on psi with a slope equal to the full formal charge of the peptides. However, the partition constant levels off at higher psi, suggesting departure from the ideal limiting behavior.

Amino Acid Sequence↗
PubMed1995

Interaction of the isolated transmembrane domain of diphtheria toxin with membranes.

Insertion of diphtheria toxin's T (transmembrane) domain into the endosomal membrane under acidic conditions is known to promote translocation of its catalytic domain across the membrane and into the cytosol. The T domain, a cysteine-free bundle of alpha-helices, was expressed as a discrete protein in Escherichia coli and purified. The isolated domain was stable and largely monomeric at pH 8.0. Like the holotoxin it bound the hydrophobic fluorophore, 2-p-toluidinylnaphthalene 6-sulfonate, upon acidification, but the transition pH was higher than with the holotoxin (pH 5.6 vs 5.1) and broader, reflecting the absence of interdomain interactions. The domain also permeabilized large unilamellar vesicles under acidic conditions, as demonstrated by release of entrapped solutes. Mutant forms of T domain, each with a single residue replaced by cysteine, were derivatized with a thiol-reactive nitroxide-containing spin label and analyzed by electron paramagnetic resonance (EPR). EPR spectra and solvent accessibilities of the labels at pH 8.0 were consistent with the environments predicted from the toxin's crystallographic structure. Acidification in the presence of large unilamellar vesicles caused a nitroxide label at position 332 on helix TH8 to move from a buried site in the water soluble state to a lipid-exposed surface site at a depth of approximately 15 A within the bilayer. This is consistent with the concept that the TH8-TH9 helix pair inserts into the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗