A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies.
AuthorsRatner V,Kahana E,Eichler M,Haas E
JournalBioconjugate chemistry
PubMed ID12236801
Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state ... More
Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation.
AuthorsBures EJ, Hui JO, Young Y, Chow DT, Katta V, Rohde MF, Zeni L, Rosenfeld RD, Stark KL, Haniu M
JournalBiochemistry
PubMed ID9724530
The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical ... More
Hydrogen exchange/electrospray ionization mass spectrometry studies of structural features of proteins and protein/protein interactions.
AuthorsEhring H
JournalAnal Biochem
PubMed ID10036128
'The rate at which amide hydrogens located at the peptide backbone in protein/protein complexes undergo hydrogen/deuterium exchange is highly dependent on whether the amide groups participate in binding. Here, a new mass spectrometric method is presented in which this effect is utilized for the characterization of protein/ligand binding sites. The ... More
A synthetic lipopolysaccharide-binding peptide based on amino acids 27-39 of serum amyloid P component inhibits lipopolysaccharide-induced responses in human blood.
Authorsde Haas CJ, van der Tol ME, Van Kessel KP, Verhoef J, Van Strijp JA
JournalJ Immunol
PubMed ID9759883
'LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using ... More
Mass spectrometric characterization of transferrins and their fragments derived by reduction of disulfide bonds.
AuthorsThevis M, Loo RR, Loo JA
JournalJ Am Soc Mass Spectrom
PubMed ID12781465
'Mass spectrometry, proteomics, and protein chemistry methods are used to characterize the cleavage products of 79 kDa transferrin proteins induced by iron-catalyzed oxidation, including a novel C-terminal polypeptide released upon disulfide reduction. Top-down electrospray ionization tandem mass spectrometry (ESI-MS/MS) of intact multiply-charged transferrin from a variety of species (human, bovine, ... More
Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor.
AuthorsKatsumi A, Tuley EA, Bodó I, Sadler JE
JournalJ Biol Chem
PubMed ID10831592
'von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between ... More
Pyrene excimer fluorescence: a spatially sensitive probe to monitor lipid-induced helical rearrangement of apolipophorin III.
AuthorsSahoo D, Narayanaswami V, Kay CM, Ryan RO
JournalBiochemistry
PubMed ID10828977
'Manduca sexta apolipophorin III (apoLp-III), an 18-kDa, monomeric, insect hemolymph apolipoprotein, is comprised of five amphipathic alpha-helices arranged as a globular bundle in the lipid-free state. Upon lipid binding, it is postulated that the bundle opens, exposing a continuous hydrophobic surface which becomes available for lipid interaction. To investigate lipid ... More
Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.
AuthorsBlanchoin L, Pollard TD
JournalJ Biol Chem
PubMed ID10336448
'We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle ... More
Luminescence resonance energy transfer measurements in myosin.
AuthorsBurmeister Getz E, Cooke R, Selvin PR
JournalBiophys J
PubMed ID9591671
'Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), ... More
Delivery of oligonucleotides into mammalian cells by anionic peptides: comparison between monomeric and dimeric peptides.
AuthorsFreulon I, Roche AC, Monsigny M, Mayer R
JournalBiochem J
PubMed ID11237872
'The use of antisense oligonucleotides as putative therapeutic agents is limited by their poor delivery into the cytosol and/or the nucleus because they are not able to efficiently cross lipid bilayers. To circumvent this pitfall, anionic amphipathic peptides derived from the influenza virus fusogenic peptide have been used to destabilize ... More
High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane.
AuthorsMansoor SE, Farrens DL
JournalBiochemistry
PubMed ID15260485
'We present a site-directed fluorescence labeling (SDFL) study of 25 different T4 lysozyme protein samples labeled with the thiol-cleavable fluorophore, (2-pyridyl)dithiobimane (PDT-Bimane). Our results demonstrate PDT-Bimane can be used in cysteine-scanning studies to detect protein secondary structure, and to map proximity between sites in proteins by monitoring tryptophan quenching of ... More
Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers.
AuthorsChen X, Wolfgang DE, Sampson NS
JournalBiochemistry
PubMed ID11063575
'To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant ... More
In situ reduction suitable for matrix-assisted laser desorption/ionization and liquid secondary ionization using tris(2-carboxyethyl)phosphine.
AuthorsFischer WH, Rivier JE, Craig AG
JournalRapid Commun Mass Spectrom
PubMed ID8481542
'The suitability of the hydrochloride salt of tris(2-carboxyethyl)phosphine (TCEP) for in situ reduction with matrix-assisted laser desorption/ionization (MALDI) and liquid secondary ionization (LSI) mass spectrometry is evaluated. TCEP can be used to irreversibly reduce organic disulfides to thiols in water and is active at an acidic pH. We found that ... More
Fluorescence anisotropy assay for proteolysis of specifically labeled fusion proteins.
AuthorsBlommel PG, Fox BG
JournalAnal Biochem
PubMed ID15582561
'A cloning method and plasmid vectors that permit fluorescence-anisotropy-based measurement of proteolysis are reported. The recombinant protein substrates produced by this method contain a tetracysteine motif that can be site-specifically labeled with bis-arsenical fluorophore [Science 281 (1998) 269]. Six protein substrates with an N-terminal fusion of the tetracysteine motif and ... More
Attachment of peptide building blocks to proteins through tyrosine bioconjugation.
AuthorsRomanini DW, Francis MB,
JournalBioconjug Chem
PubMed ID18069779
'Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues ... More
Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins.
AuthorsPutilina T, Skouri-Panet F, Prat K, Lubsen NH, Tardieu A
JournalJ Biol Chem
PubMed ID12562766
'The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for ... More
Identification of transthyretin variants by sequential proteomic and genomic analysis.
AuthorsBergen HR, Zeldenrust SR, Butz ML, Snow DS, Dyck PJ, Dyck PJ, Klein CJ, O'Brien JF, Thibodeau SN, Muddiman DC
JournalClin Chem
PubMed ID15217993
'BACKGROUND: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with ... More
Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme.
AuthorsBrohawn SG, Miksa IR, Thorpe C
JournalBiochemistry
PubMed ID12974644
'Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is ... More
Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives.
AuthorsVila A, Tallman KA, Jacobs AT, Liebler DC, Porter NA, Marnett LJ,
JournalChem Res Toxicol
PubMed ID18232660
'Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic alpha,beta-unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and ... More
Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments.
AuthorsAllen MW, Urbauer RJ, Zaidi A, Williams TD, Urbauer JL, Johnson CK
JournalAnal Biochem
PubMed ID14751262
'We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring ... More
Characterization of apolipoprotein A-I structure using a cysteine-specific fluorescence probe.
AuthorsTricerri MA, Behling Agree AK, Sanchez SA, Jonas A
JournalBiochemistry
PubMed ID11087425
'Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created and expressed in Escherichia coli systems. Specific labeling with the thiol-reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), was used to study the structural organization and dynamic properties of the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound ... More
Two-color labeling of temporally defined protein populations in mammalian cells.
AuthorsBeatty KE, Tirrell DA,
JournalBioorg Med Chem Lett
PubMed ID18774715
'The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in ... More
FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry.
'As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. ... More
Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil.
AuthorsBhattacharyya RP, Sosnick TR
JournalBiochemistry
PubMed ID10029555
'We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2'', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, ... More
A major G protein alpha O isoform in bovine brain is deamidated at Asn346 and Asn347, residues involved in receptor coupling.
'The structural differences between two major forms of the alpha subunit of the heterotrimeric G protein GO were found to be due to deamidation of either of two Asn residues near the C-terminus of the proteins, in a region involved in receptor recognition. GO is the most abundant heterotrimeric G ... More
Determination of the disulfide bond arrangement of Newcastle disease virus hemagglutinin neuraminidase. Correlation with a beta-sheet propeller structural fold predicted for paramyxoviridae attachment proteins.
AuthorsPitt JJ, Da Silva E, Gorman JJ
JournalJ Biol Chem
PubMed ID10692451
'Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by ... More
Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells.
AuthorsJessop CE, Bulleid NJ
JournalJ Biol Chem
PubMed ID15507438
'The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be ... More
Dual nanomolar and picomolar Zn(II) binding properties of metallothionein.
AuthorsKrezel A, Maret W,
JournalJ Am Chem Soc
PubMed ID17696343
'Each of the seven Zn(II) ions in the Zn(3)S(9) and Zn(4)S(11) clusters of human metallothionein is in a tetrathiolate coordination environment. Yet analysis of Zn(II) association with thionein, the apoprotein, and analysis of Zn(II) dissociation from metallothionein using the fluorescent chelating agents FluoZin-3 and RhodZin-3 reveal at least three classes ... More
Key residues characteristic of GATA N-fingers are recognized by FOG.
AuthorsFox AH, Kowalski K, King GF, Mackay JP, Crossley M
JournalJ Biol Chem
PubMed ID9837943
'Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. We have investigated the interaction between ... More
Both rotor and stator subunits are necessary for efficient binding of F1 to F0 in functionally assembled Escherichia coli ATP synthase.
'In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies ... More
Optimized conditions to couple two water-soluble biomolecules through alkylamine thiolation and thioetherification.
AuthorsMeunier L, Bourgerie S, Mayer R, Roche AC, Monsigny M
JournalBioconjug Chem
PubMed ID10077469
'A simple method for introducing, in buffered saline, a reactive sulfhydryl group on water-soluble molecules bearing an alkyl-amino group is described. This method is based on the use of two water-soluble reagents: 2-iminothiolane and 6,6''-dithiodinicotinic acid. The first one is open upon reaction with an amino group, and the generated ... 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
Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization.
AuthorsVasiliskov AV, Timofeev EN, Surzhikov SA, Drobyshev AL, Shick VV, Mirzabekov AD
JournalBiotechniques
PubMed ID10489618
'The manufacturing of microchips containing oligonucleotides and proteins immobilized within gel pads, ranging in size from 10 x 10 to 100 x 100 microns, is described. The microchips are produced by photo- or persulfate-induced copolymerization of unsaturated derivatives of biomolecules with acrylamide-bisacrylamide mixture. Oligonucleotides containing 5''-allyl or 5''-butenediol units were ... More
Optical properties and application of a reactive and bioreducible thiol-containing tetramethylrhodamine dimer.
'Thiolated dimeric tetramethylrhodamine (TAMRA) was synthesized in a straightforward procedure utilizing commercially available 5(6)-succinimidyl TAMRA and cystamine hydrochloride. The thiol-containing TAMRA dimer displayed distinct spectral properties in reduced and oxidized forms; covalent dimer formation produced greater effects on the spectral properties than previously reported for noncovalent TAMRA dimers or dimers ... More
Dual-targeted labeling of proteins using cysteine and selenomethionine residues.
AuthorsLang S, Spratt DE, Guillemette JG, Palmer M
JournalAnal Biochem
PubMed ID15950913
'A new strategy for dual site-selective labeling of proteins that uses metabolically incorporated selenomethionine as a target for covalent modification by iodoacetamide derivatives, forming selenonium salts, is described. In the absence of free cysteine, labeling is specific and efficient. Dual-targeted labeling of a protein can be achieved with combinations of ... More
A fluorescent radioiodinated oligonucleotidic photoaffinity probe for protein labeling: synthesis and photolabeling of thrombin.
AuthorsBerens C, Courtoy PJ, Sonveaux E
JournalBioconjug Chem
PubMed ID9893964
'To study the interactions between oligonucleotides and proteins, an original photoaffinity radiolabeling probe has been synthesized. Starting with a 5''-pyridyldithio-3''-amino-oligonucleotide, the photophore benzophenone was first coupled to the 3'' end, through acylation by an activated ester of benzoylbenzoic acid. A fluorescein molecule was grafted by alkylation of the free 5''-SH. ... More
Quantitation of hydrogen peroxide using tris(2-carboxyethyl)phosphine.
AuthorsHan J, Yen S, Han G, Han P
JournalAnal Biochem
PubMed ID8742092
Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.
AuthorsWu P, Shui W, Carlson BL, Hu N, Rabuka D, Lee J, Bertozzi CR,
JournalProc Natl Acad Sci U S A
PubMed ID19202059
'The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the ... More
Interaction of actin monomers with Acanthamoeba actophorin (ADF/cofilin) and profilin.
AuthorsBlanchoin L, Pollard TD
JournalJ Biol Chem
PubMed ID9737968
'Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the ... More
Plasma total homocysteine quantification: an improvement of the classical high-performance liquid chromatographic method with fluorescence detection of the thiol-SBD derivatives.
AuthorsGarcia AJ, Apitz-Castro R
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12361751
'A rapid, shorter, isocratic high-performance liquid chromatographic method is described for the determination of plasma total homocysteine. In this method the sample preparation was modified for reduction of the time of the thiolic reduction from 30 at room temperature to 10 min at 37 degrees C with tris-(2-carboxyethyl) phosphine (TCEP), ... More
A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA.
AuthorsDodge A, Turcatti G, Lawrence I, de Rooij NF, Verpoorte E
JournalAnal Chem
PubMed ID15018583
'This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the ... More
Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes.
AuthorsShaw J, Rowlinson R, Nickson J, Stone T, Sweet A, Williams K, Tonge R
JournalProteomics
PubMed ID12872219
'Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further ... More
The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions.
AuthorsMcLachlin DT, Bestard JA, Dunn SD
JournalJ Biol Chem
PubMed ID9614129
'An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity ... More
Site-directed fluorescence studies of a prokaryotic ClC antiporter.
AuthorsBell SP, Curran PK, Choi S, Mindell JA
JournalBiochemistry
PubMed ID16734414
'Channels and transporters of the ClC family serve a variety of physiological functions. Understanding of their gating and transport mechanisms remains incomplete, with disagreement over the extent of protein conformational change involved. Using site-directed fluorescence labeling, we probe ClC-ec1, a prokaryotic ClC, for transport-related structural rearrangements. We specifically label cysteines ... More
Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms.
AuthorsSmith JJ, Conrad DW, Cuneo MJ, Hellinga HW
JournalProtein Sci
PubMed ID15576565
'Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We ... More
Novel role for the NMDA receptor redox modulatory site in the pathophysiology of seizures.
AuthorsSanchez RM, Wang C, Gardner G, Orlando L, Tauck DL, Rosenberg PA, Aizenman E, Jensen FE
JournalJ Neurosci
PubMed ID10704515
'Redox-active compounds modulate NMDA receptors (NMDARs) such that reduction of NMDAR redox sites increases, and oxidation decreases, NMDAR-mediated activity. Because NMDARs contribute to the pathophysiology of seizures, redox-active compounds also may modulate seizure activity. We report that the oxidant 5, 5''-dithio-bis(2-nitrobenzoic acid) (DTNB) and the redox cofactor pyrroloquinoline quinone (PQQ) ... More
Dual-labeled glucose binding protein for ratiometric measurements of glucose.
AuthorsGe X, Tolosa L, Rao G
JournalAnal Chem
PubMed ID14987097
'Highly sensitive glucose monitoring has potential applications in conditions where the glucose levels are below the detection limit of currently available technology. Examples include bioprocess monitoring of bacterial cultures and measurement of minute amounts of human interstitial fluid extracted by iontophoresis. Here we describe a ratiometric glucose sensor capable of ... More
Dynamics of equilibrium structural fluctuations of apomyoglobin measured by fluorescence correlation spectroscopy.
AuthorsChen H, Rhoades E, Butler JS, Loh SN, Webb WW
JournalProc Natl Acad Sci U S A
PubMed ID17556539
'The spectra of equilibrium chain conformation fluctuations of apomyoglobin (apoMb) as a function of folding, from the acid-denatured state at pH 2.6 through the stable molten globule state pH approximately 4.1 to the folded state at pH 6.3, are reported, as measured by fluorescence correlation spectroscopy. The conformational fluctuations, which ... More
Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization.
AuthorsHarper SL, Begg GE, Speicher DW
JournalBiochemistry
PubMed ID11502188
'Human erythrocyte spectrin is an antiparallel heterodimer comprised of a 280 kDa alpha subunit and a 246 kDa beta subunit which further associates into tetramers in the red cell membrane cytoskeleton. Lateral association of the flexible rodlike monomers involves a multiple-step process that is initiated by a high affinity association ... More
Solid-phase biotinylation of antibodies.
AuthorsStrachan E, Mallia AK, Cox JM, Antharavally B, Desai S, Sykaluk L, O'Sullivan V, Bell PA
JournalJ Mol Recognit
PubMed ID15137036
'Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with ... More
An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations.
AuthorsHansen RE, Winther JR,
JournalAnal Biochem
PubMed ID19664585
'This review outlines the basic issues to consider when dealing with biochemical and cellular aspects of thiol–disulfide chemistry. The overall focus is on practical aspects, including typical biochemical experimental conditions and caveats to consider in interpreting results. Reagents for thiol derivatization and disulfide reduction are evaluated and compared, and we ... More
Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis.
'Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine ... More
Determination of the disulfide structure of sillucin, a highly knotted, cysteine-rich peptide, by cyanylation/cleavage mass mapping.
AuthorsQi J, Wu J, Somkuti GA, Watson JT
JournalBiochemistry
PubMed ID11294620
'The disulfide structure of sillucin, a highly knotted, cysteine-rich, antimicrobial peptide, isolated from Rhizomucor pusillus, has been determined to be Cys2--Cys7, Cys12--Cys24, Cys13--Cys30, and Cys14--Cys21 by disulfide mass mapping based on partial reduction and CN-induced cleavage enabled by cyanylation. The denatured 30-residue peptide was subjected to partial reduction by tris(2-carboxyethyl)phosphine ... More
Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging.
AuthorsCai W, Chen X,
JournalNat Protoc
PubMed ID18193025
'To take full advantage of the unique optical properties of quantum dots (QDs) and expedite future near-infrared fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably directed to a specific organ or disease site after systemic administration. Recently, we reported the use of peptide-conjugated QDs for non-invasive ... More
An unusual cleavage reaction of a peptide observed during dithiotreitol and tris(2-carboxyethyl)phosphine reduction: application to sequencing of HpTx2 spider toxin using nanospray tandem mass spectrometry.
AuthorsLegros C, Célérier ML, Guette C
JournalRapid Commun Mass Spectrom
PubMed ID15174186
'A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) ... More
Cleavage of disulfide bonds leads to inactivation and degradation of the type IIa, but not type IIb sodium phosphate cotransporter expressed in Xenopus laevis oocytes.
AuthorsLambert G, Traebert M, Biber J, Murer H
JournalJ Membr Biol
PubMed ID10926679
'Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter (rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/P(i)-cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with ... More
Preparation of 2-nitro-5-thiobenzoic acid using immobilized Tris(2-carboxyethyl)phosphine.
AuthorsHan J, Clark C, Han G, Chu TC, Han P
JournalAnal Biochem
PubMed ID10075833
Reaction of Tris(2-carboxyethyl)phosphine (TCEP) with maleimide and alpha-haloacyl groups: anomalous elution of TCEP by gel filtration.
AuthorsShafer DE, Inman JK, Lees A
JournalAnal Biochem
PubMed ID10860517
Novel reductant for determination of total plasma homocysteine.
AuthorsGilfix BM, Blank DW, Rosenblatt DS
JournalClin Chem
PubMed ID9105275
Use of tris(2-carboxyethyl)phosphine for quantitation of iodine and iodate.
AuthorsHan J, Payne V, Yen S, Han G, Han P
JournalAnal Biochem
PubMed ID8923979
An improved monobromobimane assay for glutathione utilizing tris- (2-carboxyethyl)phosphine as the reductant.
Reductive dehalogenation of monobromobimane by tris(2-carboxyethyl)phosphine.
AuthorsGraham DE, Harich KC, White RH,
JournalAnal Biochem
PubMed ID12814640
We demonstrate that the primary fluorescent product of the TCEP reaction with monobromobimane is the 9,10-dioxa-syn-(methyl,methyl)(methyl,methyl)bimane, formed by an unusual nucleophilic attack of the phosphine on the monobromobimane bromine atom. ... More
Mild and chemoselective peptide-bond cleavage of peptides and proteins at azido homoalanine.
AuthorsBack JW, David O, Kramer G, Masson G, Kasper PT, de Koning LJ, de Jong L, van Maarseveen JH, de Koster CG
JournalAngew Chem Int Ed Engl
PubMed ID16281315
The chemical cleavage of amide (peptide) bonds usually requires harsh conditions. As a result, side reactions and the lack of specificity of chemical amide-bond hydrolysis limits its scope in chemical biology and synthetic applications. Herein, we disclose our results on the selective cleavage of amide bonds in peptides and proteins ... More
Molecular basis of charge movement in voltage-gated sodium channels.
AuthorsYang N, George AL, Horn R
JournalNeuron
PubMed ID8562074
Voltage-dependent movement of a sodium channel S4 segment was examined by cysteine scanning mutagenesis and testing accessibility of the residues to hydrophilic cysteine-modifying reagents. These experiments indicate that 2 charged S4 residues move completely from an internally accessible to an externally accessible location in response to depolarization by passage through ... More
Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.
AuthorsDeRocco V, Anderson T, Piehler J, Erie DA, Weninger K,
JournalBiotechniques
PubMed ID21091445
To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to ... More
Conjugation of polyethylene glycol via a disulfide bond confers water solubility upon a peptide model of a protein transmembrane segment.
AuthorsPomroy NC, Deber CM
JournalAnal Biochem
PubMed ID10552908
The aqueous insolubility of hydrophobic peptides has presented a barrier to the structural characterization of membrane protein transmembrane domains. Since the conjugation of polyethylene glycol is known to modulate the solubility of certain proteins and peptides, we have prepared PEG-a-Cys reagent, a polyethylene glycol derivative which reacts spontaneously with Cys ... More
Novel protein transfection of primary rat cortical neurons using an antibody that penetrates living cells.
AuthorsWeisbart RH, Baldwin R, Huh B, Zack DJ, Nishimura R
JournalJ Immunol
PubMed ID10820286
An Ab-based system to deliver functional proteins into neurons was developed using the murine mAb, mAb 3E10. This was achieved by covalently conjugating catalase to the Ab so that the conjugate retained high activity for the degradation of hydrogen peroxide. Three-dimensional fluorescence microscopy was used to demonstrate penetration of the ... More
Bisarsenical labeling of HIV-1 for real-time fluorescence microscopy.
AuthorsArhel NJ, Charneau P,
JournalMethods Mol Biol
PubMed ID19020824
Imaging studies have benefited from the development of a novel technique for non-destructive labeling of proteins within living cells, based on the use of a reagent called FlAsH-EDT2, a bisarsenical derivative of fluorescein capable of binding with high affinity and specificity to a tetracysteine motif in the protein of interest. ... More
Reducing agent-mediated precipitation of high-abundance plasma proteins.
Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol ... More
Identification and quantitation of newly synthesized proteins in Escherichia coli by enrichment of azidohomoalanine-labeled peptides with diagonal chromatography.
AuthorsKramer G, Sprenger RR, Back J, Dekker HL, Nessen MA, van Maarseveen JH, de Koning LJ, Hellingwerf KJ, de Jong L, de Koster CG,
JournalMol Cell Proteomics
PubMed ID19321432
A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 ... More
The catalytic domain of insulin-degrading enzyme forms a denaturant-resistant complex with amyloid beta peptide: implications for Alzheimer disease pathogenesis.
AuthorsLlovera RE, de Tullio M, Alonso LG, Leissring MA, Kaufman SB, Roher AE, de Prat Gay G, Morelli L, Castaño EM,
JournalJ Biol Chem
PubMed ID18411275
Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid beta (Abeta) in the mammalian brain. Biochemical and genetic data support the notion that IDE may play a role in late onset Alzheimer disease (AD), and recent studies suggest an association between AD and diabetes mellitus type ... More
Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies.
The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the ... More
Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.
AuthorsGuo J, Xu N, Li Z, Zhang S, Wu J, Kim DH, Sano Marma M, Meng Q, Cao H, Li X, Shi S, Yu L, Kalachikov S, Russo JJ, Turro NJ, Ju J,
JournalProc Natl Acad Sci U S A
PubMed ID18591653
DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH ... More
Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens.
AuthorsGreengauz-Roberts O, Stöppler H, Nomura S, Yamaguchi H, Goldenring JR, Podolsky RH, Lee JR, Dynan WS,
JournalProteomics
PubMed ID15761955
Laser capture microdissection (LCM) provides the capability to isolate and analyze small numbers of cells from a specific area of a histologic section. LCM has particular value for analysis of early stage tumors, which are often small and intermixed with non-tumor tissue. It has previously been shown that a new ... More
Nitric oxide stress induces different responses but mediates comparable protein thiol protection in Bacillus subtilis and Staphylococcus aureus.
AuthorsHochgräfe F, Wolf C, Fuchs S, Liebeke M, Lalk M, Engelmann S, Hecker M,
JournalJ Bacteriol
PubMed ID18487332
The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO ... More
In vivo imaging of membrane-associated glycans in developing zebrafish.
Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with ... More
Efficient site-specific labeling of proteins via cysteines.
AuthorsKim Y, Ho SO, Gassman NR, Korlann Y, Landorf EV, Collart FR, Weiss S,
JournalBioconjug Chem
PubMed ID18275130
Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using ... More
Resolving cadherin interactions and binding cooperativity at the single-molecule level.
AuthorsZhang Y, Sivasankar S, Nelson WJ, Chu S,
JournalProc Natl Acad Sci U S A
PubMed ID19114658
The cadherin family of Ca(2+)-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell-cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 ... More
Imaging the lipidome: omega-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins.
AuthorsHannoush RN, Arenas-Ramirez N,
JournalACS Chem Biol
PubMed ID19505150
Fatty acylation or lipid modification of proteins controls their cellular activation and diverse roles in physiology. It mediates protein-protein and protein-membrane interactions and plays an important role in regulating cellular signaling pathways. Currently, there is need for visualizing lipid modifications of proteins in cells. Herein we report novel chemical probes ... More
A FRET-based method to study protein thiol oxidation in histological preparations.
AuthorsMastroberardino PG, Orr AL, Hu X, Na HM, Greenamyre JT,
JournalFree Radic Biol Med
PubMed ID18620047
Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling ... More
Copper-free click chemistry for dynamic in vivo imaging.
AuthorsBaskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, Lo A, Codelli JA, Bertozzi CR,
JournalProc Natl Acad Sci U S A
PubMed ID17942682
Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomolecules such as glycans and lipids. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living ... 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
Reversible, allosteric small-molecule inhibitors of regulator of g protein signaling proteins.
AuthorsBlazer LL, Roman DL, Chung A, Larsen MJ, Greedy BM, Husbands SM, Neubig RR,
JournalMol Pharmacol
PubMed ID20571077
Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an ... More
Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.
AuthorsHuang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall JD, Hanson GR, Stokes KC, Leopold M, Multhaup G, Goldstein LE, Scarpa RC, Saunders AJ, Lim J, Moir RD, Glabe C, Bowden EF, Masters CL, Fairlie DP, Tanzi RE, Bush AI
JournalJ Biol Chem
PubMed ID10601271
Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by ... More
Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori.
AuthorsLeal WS, Nikonova L, Peng G
JournalFEBS Lett
PubMed ID10611489
Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We ... More
Sample preparation for peptide mapping--A pharmaceutical quality-control perspective.
AuthorsLundell N, Schreitmüller T
JournalAnal Biochem
PubMed ID9887211
In quality control of therapeutic proteins peptide mapping is used for confirmation of primary structure and detection of posttranslational modifications. The demands put on the experimental procedure are therefore different than in the case of determination of an unknown protein structure. It is here recognized that a peptide-mapping method for ... More
Secretion of type III effectors into host cells in real time.
AuthorsEnninga J, Mounier J, Sansonetti P, Tran Van Nhieu G
JournalNat Methods
PubMed ID16299482
Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their ... More
Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein.
AuthorsTolosa L, Gryczynski I, Eichhorn LR, Dattelbaum JD, Castellano FN, Rao G, Lakowicz JR
JournalAnal Biochem
PubMed ID9918662
We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in ... More
Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology.
AuthorsSampathkumar SG, Li AV, Jones MB, Sun Z, Yarema KJ
JournalNat Chem Biol
PubMed ID16474386
Metabolic 'oligosaccharide engineering' methods based on N-acetyl-D-mannosamine (ManNAc) analogs allow the glycocalyx of living cells to be remodeled. Herein we report the analog Ac(5)ManNTGc (1) that enables thiols to be expressed in surface sialic acids. By locating this versatile functional group on the outer periphery of normally nonadhesive human Jurkat ... More
Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing.
AuthorsNampalli S, McDougall MG, Lavrenov K, Xiao H, Kumar S
JournalBioconjug Chem
PubMed ID12009935
Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by ... More
Coordination properties of tris(2-carboxyethyl)phosphine, a newly introduced thiol reductant, and its oxide.
AuthorsKrezel A, Latajka R, Bujacz GD, Bal W
JournalInorg Chem
PubMed ID12639134
Acid-base properties and metal-binding abilities of tris(2-carboxyethyl)phosphine (TCEP), a newly introduced thiol group protectant, were studied in solution, using potentiometry, (1)H and (31)P NMR, and UV-vis spectroscopy, and also in the solid state by X-ray diffraction. Stability constants of complexes of the P-oxide of TCEP (TCEPO) were established by potentiometry. ... More
Differential binding of traffic-related proteins to phosphatidic acid- or phosphatidylinositol (4,5)- bisphosphate-coupled affinity reagents.
AuthorsManifava M, Thuring JW, Lim ZY, Packman L, Holmes AB, Ktistakis NT
JournalJ Biol Chem
PubMed ID11124268
Phosphatidic acid (PA) is an important bioactive lipid, but its molecular targets remain unknown. To identify such targets, we have synthesized and coupled PA to an agarose-based matrix, Affi-Gel 10. Using this matrix as an affinity reagent, we have identified a substantial number of potential PA-binding proteins from brain cytosol. ... More
Implication of a novel multiprotein Dam1p complex in outer kinetochore function.
AuthorsCheeseman IM, Brew C, Wolyniak M, Desai A, Anderson S, Muster N, Yates JR, Huffaker TC, Drubin DG, Barnes G
JournalJ Cell Biol
PubMed ID11756468
Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins ... More
A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
AuthorsWu J, Gage DA, Watson JT
JournalAnal Biochem
PubMed ID8833324
A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ ionization time-of flight mass spectrometry (MALDITOF MS). This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) ... More
A novel post-translational modification involving bromination of tryptophan. Identification of the residue, L-6-bromotryptophan, in peptides from Conus imperialis and Conus radiatus venom.
We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected ... More
Peptide-based targeting of fluorophores to organelles in living cells.
AuthorsPap EH, Dansen TB, van Summeren R, Wirtz KW
JournalExp Cell Res
PubMed ID11302694
Peptides carrying organelle-specific import or retention sequences can target the fluorophore BODIPY(581/591) to the nucleus, peroxisomes, endoplasmic reticulum (ER), or the trans-Golgi network (TGN). The peroxisomal peptide contains the PTS1 sequence AKL. For targeting to the ER or TGN, the peptides carry the retention sequences KDEL and SDYQRL, respectively. A ... More
Evidence that the beta-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of abeta by zinc.
AuthorsCuajungco MP, Goldstein LE, Nunomura A, Smith MA, Lim JT, Atwood CS, Huang X, Farrag YW, Perry G, Bush AI
JournalJ Biol Chem
PubMed ID10801774
Abeta binds Zn(2+), Cu(2+), and Fe(3+) in vitro, and these metals are markedly elevated in the neocortex and especially enriched in amyloid plaque deposits of individuals with Alzheimer's disease (AD). Zn(2+) precipitates Abeta in vitro, and Cu(2+) interaction with Abeta promotes its neurotoxicity, correlating with metal reduction and the cell-free ... More
Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry.
AuthorsRhee SS, Burke DH
JournalAnal Biochem
PubMed ID14715294
We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [(N-acryloylamino)phenyl] ... More
A membrane-targeted BID BCL-2 homology 3 peptide is sufficient for high potency activation of BAX in vitro.
The multidomain pro-apoptotic proteins BAX and BAK constitute an essential gateway to mitochondrial dysfunction and programmed cell death. Among the "BCL-2 homology (BH) 3-only" members of pro-apoptotic proteins, truncated BID (tBID) has been implicated in direct BAX activation, although an explicit molecular mechanism remains elusive. We find that BID BH3 ... More