Thiol/disulfide exchange is required for membrane fusion directed by the Newcastle disease virus fusion protein.
AuthorsJain S, McGinnes LW, Morrison TG
JournalJ Virol
PubMed ID17151113
'Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought ... More
Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes.
AuthorsWordeman L, Steuer ER, Sheetz MP, Mitchison T
JournalJ Cell Biol
PubMed ID1830054
'We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. ... More
Chemical modification of rat hepatic microsomes with N-ethylmaleimide results in inactivation of both UDP-N-acetylglucosamine-dependent stimulation of glucuronidation and UDP-glucuronic acid uptake.
AuthorsIkushiro S, Emi Y, Kimura S, Iyanagi T
JournalBiochim Biophys Acta
PubMed ID10434058
'Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various ... More
Functional symmetry of UhpT, the sugar phosphate transporter of Escherichia coli.
AuthorsFann MC, Maloney PC
JournalJ Biol Chem
PubMed ID9837961
'UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate ... More
Evidence for activation of tissue factor by an allosteric disulfide bond.
'Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF ... More
Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.
AuthorsWagenknecht T, Berkowitz J, Grassucci R, Timerman AP, Fleischer S
JournalBiophys J
PubMed ID7696469
'Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a ... More
A novel topology and redox regulation of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2.
AuthorsCai X, Zhang K, Lytton J
JournalJ Biol Chem
PubMed ID12377762
'In this study we have examined the roles of endogenous cysteine residues in the rat brain K(+)-dependent Na(+)/Ca(2+) exchanger protein, NCKX2, by site-directed mutagenesis. We found that mutation of Cys-614 or Cys-666 to Ala inhibited expression of the exchanger protein in HEK-293 cells, but not in an in vitro translation ... More
Disulfide isomerization switches tissue factor from coagulation to cell signaling.
AuthorsAhamed J, Versteeg HH, Kerver M, Chen VM, Mueller BM, Hogg PJ, Ruf W
JournalProc Natl Acad Sci U S A
PubMed ID16959886
'Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF.VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys186-Cys209 ... More
Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures.
AuthorsAngelides KJ, Smith KE, Takeda M
JournalJ Cell Biol
PubMed ID2925792
'We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the ... More
Close proximity of a cytoplasmic loop of subunit a with c subunits of the ATP synthase from Escherichia coli.
AuthorsZhang D, Vik SB
JournalJ Biol Chem
PubMed ID12525480
'Interactions between subunit a and the c subunits of the Escherichia coli ATP synthase are thought to control proton translocation through the F(o) sector. In this study cysteine substitution mutagenesis was used to define the cytoplasmic ends of the first three transmembrane spans of subunit a, as judged by accessibility ... More
Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.
AuthorsSwartz DR, Moss RL, Greaser ML
JournalBiophys J
PubMed ID9199794
'Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the ... More
Transmembrane topography of the 100-kDa a subunit (Vph1p) of the yeast vacuolar proton-translocating ATPase.
AuthorsLeng XH, Nishi T, Forgac M
JournalJ Biol Chem
PubMed ID10329659
'The membrane topography of the yeast vacuolar proton-translocating ATPase a subunit (Vph1p) has been investigated using cysteine-scanning mutagenesis. A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity. Single cysteine residues were introduced at 13 sites within the Cys-less ... More
Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain.
AuthorsZiebell MR, Zhao ZG, Luo B, Luo Y, Turley EA, Prestwich GD
JournalChem Biol
PubMed ID11731299
'BACKGROUND: Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) that promotes motility, adhesion, and proliferation in mammalian cells, as mediated by cell-surface HA receptors. We sought to identify non-carbohydrate ligands that would bind to and activate cell-surface HA receptors. Such analogs could have important therapeutic uses in the treatment of cancer, ... More
Selective labeling of functional groups on membrane proteins or glycoproteins using reactive biotin derivatives and 125I-streptavidin.
AuthorsRoffman E, Meromsky L, Ben-Hur H, Bayer EA, Wilchek M
JournalBiochem Biophys Res Commun
PubMed ID3085669
'Amino groups, sulfhydryl groups or oxidation-induced aldehydes on erythrocyte membrane proteins and/or glycoproteins, were reacted with biotinyl N-hydroxysuccinimide ester (BNHS), 3-(N-maleimido-propionyl) biocytin (MPB) or biocytin hydrazide (BCHZ), respectively. The detergent-lysed biotinylated samples were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose membranes. The blot was then ... More
Identification of amino acids in the binding pocket of the human KDEL receptor.
AuthorsScheel AA, Pelham HR
JournalJ Biol Chem
PubMed ID9442098
'Retention of soluble proteins in the endoplasmic reticulum is dependent on their interaction with the KDEL (Lys-Asp-Glu-Leu) receptor in the Golgi apparatus and their subsequent retrieval back to the endoplasmic reticulum. We have studied the three-dimensional organization of the human KDEL receptor using site-directed mutagenesis and sulfhydryl-specific labeling. We have ... More
Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes.
AuthorsAnderluh G, Barlic A, Podlesek Z, Macek P, Pungercar J, Gubensek F, Zecchini ML, Serra MD, Menestrina G
JournalEur J Biochem
PubMed ID10429196
'Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in ... More
The topology of VDAC as probed by biotin modification.
AuthorsSong J, Midson C, Blachly-Dyson E, Forte M, Colombini M
JournalJ Biol Chem
PubMed ID9733730
'The outer membrane of mitochondria contains channels called VDAC (mitochondrial porin), which are formed by a single 30-kDa protein. Cysteine residues introduced by site-directed mutagenesis at sites throughout Neurospora crassa VDAC (naturally devoid of cysteine) were specifically biotinylated prior to reconstitution into planar phospholipid membranes. From previous studies, binding of ... More
N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain.
AuthorsPhelps KK, Walker RA
JournalBiochemistry
PubMed ID10451370
'N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd ... More
Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1.
AuthorsTang XB, Casey JR
JournalBiochemistry
PubMed ID10545179
'AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4''-diisothiocyanostilbene-2, 2''-disulfonate) and DNDS (4,4''-dinitrostilbene-2,2''-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the regions of AE1 that undergo conformational changes ... More
The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity.
AuthorsIwamoto T, Uehara A, Imanaga I, Shigekawa M
JournalJ Biol Chem
PubMed ID10967097
'We examined the membrane topology and functional importance of residues in regions of the Na(+)/Ca(2+) exchanger NCX1 encompassing the conserved internal alpha repeats by substituted cysteine scanning analysis and kinetic analysis of site-directed mutants. The results suggest that both the alpha-1 repeat and a region encompassing the alpha-2 repeat and ... More
Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein.
AuthorsShingledecker K, Jiang S, Paulus H
JournalArch Biochem Biophys
PubMed ID10683259
'Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice ... More
Localization of a voltage gate in connexin46 gap junction hemichannels.
AuthorsPfahnl A, Dahl G
JournalBiophys J
PubMed ID9788927
'Cysteine replacement mutagenesis has identified positions in the first transmembrane domain of connexins as contributors to the pore lining of gap junction hemichannels (Zhou et al. 1997. Biophys. J. 72:1946-1953). Oocytes expressing a mutant cx46 with a cysteine in position 35 exhibited a membrane conductance sensitive to the thiol reagent ... More
Role of sulfhydryl groups in the function of glucosidase I from mammary gland.
AuthorsPukazhenthi BS, Muniappa N, Vijay IK
JournalJ Biol Chem
PubMed ID8454617
'Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc transfer from the lipid-linked donor to the nascent polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking reagents also ... More
Fluorescence microscopy methods in the study of protein structure and function.
'As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we ... More
The avidin-biotin complex in bioanalytical applications.
AuthorsWilchek M, Bayer EA
JournalAnal Biochem
PubMed ID3044183
Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.
'Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% ... More
Functional importance and local environments of the cysteines in the tetracycline resistance protein encoded by plasmid pBR322.
AuthorsJewell JE, Orwick J, Liu J, Miller KW
JournalJ Bacteriol
PubMed ID10049405
'The properties of the cysteines in the pBR322-encoded tetracycline resistance protein have been examined. Cysteines are important but not essential for tetracycline transport activity. None of the cysteines reacted with biotin maleimide, suggesting that they are shielded from the aqueous phase or reside in a negatively charged local environment.' ... More
Second-site cleavage in sterol regulatory element-binding protein occurs at transmembrane junction as determined by cysteine panning.
AuthorsDuncan EA, Davé UP, Sakai J, Goldstein JL, Brown MS
JournalJ Biol Chem
PubMed ID9651382
'In response to sterol deprivation, two sequential proteolytic cleavages release the NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) from cell membranes. The fragments translocate to the nucleus where they activate genes involved in cholesterol and fatty acid metabolism. The SREBPs are bound to membranes in a hairpin fashion. The ... More
3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent.
AuthorsBayer EA, Zalis MG, Wilchek M
JournalAnal Biochem
PubMed ID3935007
'A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin (MPB), was synthesized and used to biotinylate various proteins via native or artificially induced sulfhydryl groups. In combination with appropriate avidin- or streptavidin-conjugated markers (i.e., fluorescent, enzyme-conjugated, electron-dense, etc.), MPB essentially constitutes a universal, multipurpose, thiol-specific probe. The reagent could be used to detect ... More
Collagenous sequence governs the trimeric assembly of collagen XII.
AuthorsMazzorana M, Cogne S, Goldschmidt D, Aubert-Foucher E
JournalJ Biol Chem
PubMed ID11375984
'A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange ... More
Disulfide exchange in domain 2 of CD4 is required for entry of HIV-1.
AuthorsMatthias LJ, Yam PT, Jiang XM, Vandegraaff N, Li P, Poumbourios P, Donoghue N, Hogg PJ
JournalNat Immunol
PubMed ID12089508
'CD4, a member of the immunoglobulin superfamily of receptors that mediates cell-cell interactions in the immune system, is the primary receptor for HIV-1. The extracellular portion of CD4 is a concatenation of four immunoglobulin-like domains, D1 to D4. The D1, D2 and D4 domains each contain a disulfide bond. We ... More
Enzymatic and biochemical probes of residues external to the translocation pathway of UhpT, the sugar phosphate carrier of Escherichia coli.
AuthorsMatos M, Fann MC, Yan RT, Maloney PC
JournalJ Biol Chem
PubMed ID8702506
'Part of the substrate translocation pathway through UhpT, the Escherichia coli sugar phosphate carrier, has been assigned to a transmembrane helix extending between residues 260 and 282. To set limits on the external portion of the pathway, we identified nearby residues fully exposed to the periplasm. In one case, we ... More
Membrane topology of the di- and tripeptide transport protein of Lactococcus lactis.
AuthorsHagting A, vd Velde J, Poolman B, Konings WN
JournalBiochemistry
PubMed ID9184160
'Transport of hydrophilic di- and tripeptides into Lactococcus lactis is mediated by a proton motive force-driven peptide transport protein (DtpT) that shares similarity with eukaryotic peptide transporters, e.g., from kidney and small intestine of rabbit, man, and rat. Hydropathy profiling in combination with the "positive inside rule" predicts for most ... More
Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods.
AuthorsCao W, Matherly LH
JournalBiochem J
PubMed ID14602046
'The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting ... More
Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186.
AuthorsPalmer M, Jursch R, Weller U, Valeva A, Hilgert K, Kehoe M, Bhakdi S
JournalJ Biol Chem
PubMed ID8505320
'Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-forming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biological activity. In this study, we have used site-directed mutagenesis to introduce cysteine residues at positions 69, 130, and 186. Each mutant was fully ... More
Measurement of protein thiols after heat shock using 3-(-N-maleimido-propionyl) biocytin labeled proteins separated by SDS-PAGE and electroluted onto nitrocellulose: thiol blotting.
AuthorsFreeman ML, Meredith MJ
JournalRadiat Res
PubMed ID2493669
'We tested the hypothesis that depletion of intracellular glutathione (GSH) during heat shock results in protein thiol oxidation, thereby increasing thermal sensitivity. Depletion of GSH was accomplished using a combination of diethylmaleate and buthionine sulfoximine and protein sulfhydryls were measured using two independent methods. Chinese hamster ovary (CHO) cells were ... More
Topology of the region surrounding Glu681 of human AE1 protein, the erythrocyte anion exchanger.
AuthorsTang XB, Fujinaga J, Kopito R, Casey JR
JournalJ Biol Chem
PubMed ID9712881
'AE1 protein transports Cl- and HCO3- across the erythrocyte membrane by an electroneutral exchange mechanism. Glu681 of human AE1 may form part of the anion translocation apparatus and the permeability barrier. We have therefore studied the structure of the sequence surrounding Glu681, using scanning cysteine mutagenesis. Residues of the Ser643 ... More
Labeling of DNA via rearrangement of S-2-aminoethyl phosphorothioates to N-(2-mercaptoethyl)phosphoramidates.
AuthorsChen M, Gothelf KV,
JournalOrg Biomol Chem
PubMed ID18292883
The reaction of phosphorothioates in DNA with 2-bromoethylammonium bromide results in S-2-aminoethyl phosphorothioates, which can rearrange to N-(2-mercaptoethyl)phosphoramidates providing a facile method for the generation of site-specific thiol labeling of DNA sequences. The applicability of this method was demonstrated by conjugation of the thiolated DNA sequence with Na-(3-maleimidylpropionyl) biocytin and ... More
Biotinylation reagents for the study of cell surface proteins.
AuthorsElia G,
JournalProteomics
PubMed ID18763706
The extraordinarily stable, non-covalent interaction between avidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for derivatization with biotin of a variety of molecules (in particular, proteins) have been introduced, in order to allow their efficient recovery, immobilization and detection with avidin-based reagents. ... More
S-glutathionylation impairs signal transducer and activator of transcription 3 activation and signaling.
AuthorsXie Y, Kole S, Precht P, Pazin MJ, Bernier M,
JournalEndocrinology
PubMed ID18988672
S-glutathionylation is a physiological, reversible protein modification of cysteine residues with glutathione in response to mild oxidative stress. Because the key cell growth regulator signal transducer and activator of transcription (STAT) 3 is particularly susceptible to redox regulation, we hypothesized that oxidative modification of cysteine residues of STAT3 by S-glutathionylation ... More
Reversibility of covalent electrophile-protein adducts and chemical toxicity.
AuthorsLin D, Saleh S, Liebler DC,
JournalChem Res Toxicol
PubMed ID19548357
The biotin-tagged electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) have been used as model electrophile probes in complex proteomes to identify protein targets associated with chemical toxicity. Whereas IAB activates stress signaling and apoptosis in HEK293 cells, BMCC does not. Cysteine Michael adducts formed from BMCC and nonbiotinylated analogues rapidly disappeared ... More
Three possible disulfides in the acetylcholine receptor alpha-subunit.
AuthorsMosckovitz R, Gershoni JM
JournalJ Biol Chem
PubMed ID3121617
The cysteinyl residues of the acetylcholine receptor alpha-subunit of Torpedo californica were analyzed. All seven cysteines could be accounted for. Three possible disulfide bridges and one unpaired cysteine were indicated. The disulfide linkages were as follows: Cys128 to Cys142; Cys192 to Cys193; Cys412 to Cys418 (Cys222 is unpaired). The identification ... More
Organization in the membrane of the N-terminal proton-translocating domain of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli.
AuthorsGlavas NA, Hou C, Bragg PD
JournalBiochem Biophys Res Commun
PubMed ID7669043
The proton-translocating transmembrane pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta. The beta subunit has several membrane-spanning segments in the N-terminal region followed by a cytosolic C-terminal domain bearing a binding site for NADP(H). The N-terminal region contains at least one residue ... More
Immobilization of sugar-non-specific nucleases by utilizing the streptavidin--biotin interaction.
AuthorsGast FU, Franke I, Meiss G, Pingoud A
JournalJ Biotechnol
PubMed ID11278037
Due to their high enzymatic activity, the sugar-non-specific endonucleases from Serratia marcescens and Anabaena can be used for a number of applications, such as the removal of contaminating genetic material from biological preparations, footprinting studies, and the determination of nucleic acids in biochemical samples. These methods would benefit from immobilized ... More
A novel labeling approach supports the five-transmembrane model of subunit a of the Escherichia coli ATP synthase.
AuthorsWada T, Long JC, Zhang D, Vik SB
JournalJ Biol Chem
PubMed ID10358096
Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B ... More
Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID7559432
The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992). To test ... More
Protein disulfide isomerase and sulfhydryl-dependent pathways in platelet activation.
AuthorsEssex DW, Li M, Miller A, Feinman RD
JournalBiochemistry
PubMed ID11352743
The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation ... More
Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering.
AuthorsMiller MA, Geren L, Han GW, Saunders A, Beasley J, Pielak GJ, Durham B, Millett F, Kraut J
JournalBiochemistry
PubMed ID8547245
A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys ... More
Pyrrolidine dithiocarbamate (PDTC) is a thiol compound widely used to study the activation of redox-sensitive transcription factors. Although normally used as an antioxidant, PDTC has been shown to exert pro-oxidant activity on proteins both in vitro and in vivo. Because p53 redox status has been shown to alter its DNA ... More
Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID9829963
Human P-glycoprotein is synthesized in HEK 293 cells as two major products: the 150-kDa core-glycosylated intermediate and the 170-kDa mature proteins. The 150- and 170-kDa proteins were not detected in mutants such as G341C. The major protein in this mutant was a 130-kDa proteolytic degradation product. This result suggested that ... More
Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues.
AuthorsLong JC, Wang S, Vik SB
JournalJ Biol Chem
PubMed ID9632682
The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, ... More
Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons.
AuthorsTakeda S, Okabe S, Funakoshi T, Hirokawa N
JournalJ Cell Biol
PubMed ID7929561
Neurofilaments (NFs) are composed of triplet proteins, NF-H, NF-M, and NF-L. To understand the dynamics of NFs in vivo, we studied the dynamics of NF-H and compared them to those of NF-L, using the combination of microinjection technique and fluorescence recovery after photobleaching. In the case of NF-L protein, the ... More
Covalent binding of peptides to the N-terminal hydrophobic region of cardiac troponin C has limited effects on function.
AuthorsLin X, Dotson DG, Putkey JA
JournalJ Biol Chem
PubMed ID8550567
Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide ... More
A small catalytic RNA motif with Diels-Alderase activity.
AuthorsSeelig B, Jäschke A
JournalChem Biol
PubMed ID10074465
BACKGROUND: The 'RNA world' hypothesis requires that RNA be able to catalyze a wide variety of chemical reactions. In vitro selection from combinatorial RNA libraries has been used to identify several catalytic activities, most of which have resulted in a self-modification of RNA at one of its constituents. The formation ... More
Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I.
AuthorsJin P, Sun J, Chitnis PR
JournalBiochim Biophys Acta
PubMed ID10076011
PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography ... More
Unidirectional reconstitution and characterization of purified Na+/proline transporter of Escherichia coli.
AuthorsJung H, Tebbe S, Schmid R, Jung K
JournalBiochemistry
PubMed ID9693004
A simple approach for large-scale purification and unidirectional reconstitution of the Na+/proline transporter of Escherichia coli (PutP) is described. The procedure is based on the insertion of a highly polar peptide composed of 17 amino acids including a 6His tag at the C-terminus of the transporter. Purification of the hybrid ... More
Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli.
AuthorsSun J, Wu J, Carrasco N, Kaback HR
JournalBiochemistry
PubMed ID8547282
Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., ... More
Abnormal thiol reactivity of tropomyosin in essential hypertension and its association with abnormal sodium-lithium countertransport kinetics.
AuthorsWatkins SL, West IC, Wilkinson R, Thomas TH
JournalJ Hypertens
PubMed ID11288819
OBJECTIVES: To identify a thiol protein that is abnormal in a subgroup of essential hypertensive (EHT) patients who have a strong family history of hypertension and cardiovascular disease and have a low Km of erythrocyte Na/Li countertransport (CT). METHODS: To detect biotin maleimide labelling of a key thiol protein to ... More
A complete mechanism for steady-state oxidation of yeast cytochrome c by yeast cytochrome c peroxidase.
AuthorsMiller MA
JournalBiochemistry
PubMed ID8961942
Steady-state oxidation of yeast cytochrome c (yCc) was monitored as a function of ionic strength (mu) for mutants of a cloned cytochrome c peroxidase [CcP(MI)]. The data are best interpreted in the context of a two binding site model, where the affinity of the two sites for yCc differs by ... More
Oxidizing side of the cyanobacterial photosystem I. Evidence for interaction between the electron donor proteins and a luminal surface helix of the PsaB subunit.
AuthorsSun J, Xu W, Hervás M, Navarro JA, Rosa MA, Chitnis PR
JournalJ Biol Chem
PubMed ID10383406
Photosystem I (PSI) interacts with plastocyanin or cytochrome c6 on the luminal side. To identify sites of interaction between plastocyanin/cytochrome c6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein from Synechocystis sp. PCC 6803. The eight mutant strains differed in their ... More
Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin.
Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our ... More
Binding of the cGMP-gated channel to the Na/Ca-K exchanger in rod photoreceptors.
AuthorsSchwarzer A, Schauf H, Bauer PJ
JournalJ Biol Chem
PubMed ID10788457
The intracellular Ca(2+) concentration in rod outer segments of vertebrate photoreceptors is controlled by Ca(2+) influx through cGMP-gated channels and by Ca(2+) efflux driven by Na/Ca-K exchangers. Previously, we suggested that channel and exchanger are associated (Bauer, P. J., and Drechsler, M. (1992) J. Physiol. (Lond. ) 451, 109-131). This ... More
The beta subunit of the high conductance calcium-activated potassium channel. Identification of residues involved in charybdotoxin binding.
AuthorsHanner M, Vianna-Jorge R, Kamassah A, Schmalhofer WA, Knaus HG, Kaczorowski GJ, Garcia ML
JournalJ Biol Chem
PubMed ID9632689
Coexpression of alpha and beta subunits of the high conductance Ca2+-activated K+ (maxi-K) channel leads to a 50-fold increase in the affinity for 125I-charybdotoxin (125I-ChTX) as compared with when the alpha subunit is expressed alone (Hanner, M., Schmalhofer, W. A., Munujos, P., Knaus, H.-G., Kaczorowski, G. J., and Garcia, M. ... More
Transmembrane segment (TMS) VIII of the Na(+)/Citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane.
Authorsvan Geest M, Lolkema JS
JournalJ Biol Chem
PubMed ID10514443
The amino acid sequence of the sodium ion-dependent citrate transporter CitS of K. pneumoniae contains 12 hydrophobic stretches that could form membrane-spanning segments. A previous analysis of the membrane topology in Escherichia coli using the PhoA gene fusion technique indicated that only nine of these hydrophobic segments span the membrane, ... More
Localization of cytosolically oriented maleimide-reactive domain of vacuolar H(+)-pyrophosphatase.
AuthorsZhen RG, Kim EJ, Rea PA
JournalJ Biol Chem
PubMed ID8083239
The vacuolar H(+)-pyrophosphatase (V-PPase) of plant cells is subject to substrate (Mg2PPi)-protectable, free PPi-potentiated irreversible inhibition by the sulfhydryl reagent N-ethylmaleimide (NEM). Inhibition by NEM approximates pseudo-first order kinetics and double-log plots of the first order rate constant for inactivation versus NEM concentration yield a straight line relationship with a ... More
A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase.
AuthorsNewton GL, Av-Gay Y, Fahey RC
JournalBiochemistry
PubMed ID10978158
Mycothiol, 1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH), is composed of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes. When Mycobacterium smegmatis was treated with the alkylating agent monobromobimane (mBBr), the cellular mycothiol was converted to its bimane derivative (MSmB). The latter was rapidly cleaved to produce ... More
Unidirectional reconstitution into detergent-destabilized liposomes of the purified lactose transport system of Streptococcus thermophilus.
The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S. thermophilus, respectively. In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter. Also a LacS ... More
The transport activity of the Na+-Ca2+ exchanger NCX1 expressed in HEK 293 cells is sensitive to covalent modification of intracellular cysteine residues by sulfhydryl reagents.
AuthorsRen X, Kasir J, Rahamimoff H
JournalJ Biol Chem
PubMed ID11134012
Membrane permeable N-ethylmaleimide (NEM) and (2-aminoethyl)methanethiosulfonatehydrobromide (MTSEA) inhibited the rat brain Na(+)-Ca(2+) exchanger RBE-2 (NCX1.5) expressed in HEK 293 cells in a dose dependent manner. 50% inhibition was obtained at 1 mm MTSEA and 1.65 mm NEM. External application of membrane impermeable [2-(trimethylammonium) ethyl]methanethiosulfonatebromide (MTSET) and sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES) did not ... More
Mutations of Arg440 and Gly455/Gly456 oppositely change pH sensing of Na+/H+ exchanger 1.
AuthorsWakabayashi S, Hisamitsu T, Pang T, Shigekawa M
JournalJ Biol Chem
PubMed ID12562776
To identify important amino acid residues involved in intracellular pH (pH(i)) sensing of Na(+)/H(+) exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of ... More
Cysteine scanning mutagenesis of the noncatalytic nucleotide binding site of the yeast V-ATPase.
AuthorsVasilyeva E, Liu Q, MacLeod KJ, Baleja JD, Forgac M
JournalJ Biol Chem
PubMed ID10617613
To investigate residues involved in the formation of the noncatalytic nucleotide binding sites of the vacuolar proton-translocating adenosine triphosphatase (V-ATPase), cysteine scanning mutagenesis of the VMA2 gene that encodes the B subunit in yeast was performed. Replacement of the single endogenous cysteine residue at position 188 gave rise to a ... More
Close approximation of putative alpha -helices II, IV, VII, X, and XI in the translocation pathway of the lactose transport protein of Streptococcus thermophilus.
AuthorsVeenhoff LM, Geertsma ER, Knol J, Poolman B
JournalJ Biol Chem
PubMed ID10816556
The lactose transport protein (LacS) of Streptococcus thermophilus belongs to a family of transporters in which putative alpha-helices II and IV have been implicated in cation binding and the coupled transport of the substrate and the cation. Here, the analysis of site-directed mutants shows that a positive and negative charge ... More
Novel biotinylated phenylarsonous acids as bifunctional reagents for spatially close thiols: studies on reduced antibodies and the agonist binding site of reduced Torpedo nicotinic receptors.
AuthorsMoaddel R, Sharma A, Huseni T, Jones GS, Hanson RN, Loring RH
JournalBioconjug Chem
PubMed ID10411461
We synthesized three novel organoarsenicals as prototype bifunctional reagents for spatially close thiols, N-(4-arsenosophenyl) hexahydro-2-oxo-(3aS,4S,6aR)-1H-thieno[3, 4-d]imidazole-4-pentamide (1), 2-[4-[(4-arsenosophenyl)amino]-1, 4-dioxobutyl] hydrazide, (3aS,4S,6aR)-hexahydro-2-oxo- 1H-thieno[3, 4-d] imidazole-4-pentanoic acid (2), and [4-[[12-[[5-[(3aS,4S, 6aR)-hexahydro-2-oxo-1H-thieno[3, 4-d]imidazol-4-yl]-1-oxopentyl]amino]-1-oxododecyl]amino]phe nyl]-arso nous acid (3) containing both biotin and arsenic with intervening varying length spacers extending from 2 to 15 A ... More
Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus.
AuthorsAsai Y, Shoji T, Kawagishi I, Homma M
JournalJ Bacteriol
PubMed ID10648526
The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, and motY, in Vibrio alginolyticus. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ... More
Membrane topology of a cysteine-less mutant of human P-glycoprotein.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID7822320
A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect ... More
Generation of angiostatin by reduction and proteolysis of plasmin. Catalysis by a plasmin reductase secreted by cultured cells.
AuthorsStathakis P, Fitzgerald M, Matthias LJ, Chesterman CN, Hogg PJ
JournalJ Biol Chem
PubMed ID9252380
Extracellular manipulation of protein disulfide bonds has been implied in diverse biological processes, including penetration of viruses and endotoxin into cells and activation of certain cytokine receptors. We now demonstrate reduction of one or more disulfide bonds in the serine proteinase, plasmin, by a reductase secreted by Chinese hamster ovary ... More
Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.
AuthorsCai K, Itoh Y, Khorana HG
JournalProc Natl Acad Sci U S A
PubMed ID11320237
Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic ... More
Selective labeling of sulfhydryls and disulfides on blot transfers using avidin-biotin technology: studies on purified proteins and erythrocyte membranes.
AuthorsBayer EA, Safars M, Wilchek M
JournalAnal Biochem
PubMed ID2437828
Various strategies for the use of 3-(N-maleimido-propionyl) biocytin (MPB) as a general label for distinguishing between protein sulfhydryls and disulfides on blot transfers are presented. In the first approach, endogenous SH groups in proteins were labeled directly with MPB. For disulfide staining, endogenous sulfhydryls were blocked with N-ethylmaleimide, disulfides were ... More
Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site.
AuthorsMei H, Wang K, McKee S, Wang X, Waldner JL, Pielak GJ, Durham B, Millett F
JournalBiochemistry
PubMed ID8961943
A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constants kf and kd of the high-affinity complex between yeast iso-1-cytochrome c (yCc) and cytochrome c peroxidase compound I (CMPI) over a wide range of ionic strength. These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to ... More
Paradoxical effect of reagents for sulfhydryl and disulfide groups on human sperm capacitation and superoxide production.
Authorsde Lamirande E, Gagnon C
JournalFree Radic Biol Med
PubMed ID9823546
Spermatozoa must undergo capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human sperm capacitation, superoxide ... More