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
Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors.
AuthorsZheng Y, Shopes B, Holowka D, Baird B
JournalBiochemistry
PubMed ID1387320
'Dynamic conformations of two distinct immunoglobulin (Ig) isotypes, murine IgE and human IgG1, were examined with fluorescence resonance energy transfer measurements. The IgE mutant epsilon/C gamma 3* and the IgG1 mutant gamma/C gamma 3* each bind [5-(dimethylamino)naphthalen-1-yl]sulfonyl (DNS) in two identical antigen binding sites at the amino (N)-terminal ends of ... More
Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin.
AuthorsGraceffa P
JournalBiochemistry
PubMed ID9092808
'Smooth muscle caldesmon is a single polypeptide chain with its NH2- and COOH-terminal domains separated by a long alpha-helix. Caldesmon was labeled at either Cys-153 in the NH2 domain or Cys-580 in the COOH domain with a variety of fluorescence probes. Fluorescence intensity, peak position, and polarization of probes on ... More
Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.
AuthorsMoore JD, Yang J, Truant R, Kornbluth S
JournalJ Cell Biol
PubMed ID9922449
'Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear ... More
Close proximity of Cys64 and Cys140 in the delta subunit of Escherichia coli F1-ATPase.
AuthorsZiegler M, Xiao R, Penefsky HS
JournalJ Biol Chem
PubMed ID8307987
'The delta subunit of the F1-ATPase from Escherichia coli contains 2 cysteine residues, one at position 64 and the second at position 140 of the amino acid sequence. These residues were specifically labeled with sulfhydryl reagents in this study without labeling other -SH groups in the enzyme. Modification of Cys140 ... More
Role of transmembrane segment 10 in efflux mediated by the staphylococcal multidrug transport protein QacA.
AuthorsXu Z, O'Rourke BA, Skurray RA, Brown MH
JournalJ Biol Chem
PubMed ID16282328
'The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the ... More
Structural organization of chloroplast coupling factor.
AuthorsSnyder B, Hammes GG
JournalBiochemistry
PubMed ID2859887
'Fluorescence resonance energy transfer measurements have been used to construct spatial maps for the accessible sulfhydryl of the gamma subunit (dark site) and the essential tyrosine residue of the beta subunits relative to previously mapped sites on the H+-ATPase from chloroplasts. The extent of energy transfer was measured between a ... More
Regions of association between the alpha and the beta subunit of the gastric H,K-ATPase.
AuthorsMelle-Milovanovic D, Milovanovic M, Nagpal S, Sachs G, Shin JM
JournalJ Biol Chem
PubMed ID9556592
'A binding and a yeast two-hybrid analysis were carried out on the gastric H,K-ATPase to determine interactive regions of the extracytoplasmic domains of the alpha and beta subunits of this P type ATPase. Wheat germ agglutinin fractionation of fluorescein 5-maleimide-labeled tryptic fragments of detergent-solubilized H, K-ATPase showed that a fragment ... More
Streptolysin O: inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation.
AuthorsAbdel Ghani EM, Weis S, Walev I, Kehoe M, Bhakdi S, Palmer M
JournalBiochemistry
PubMed ID10563803
'Streptolysin O is a four-domain protein toxin that permeabilizes animal cell membranes. The toxin first binds as a monomer to membrane cholesterol and subsequently assembles into oligomeric transmembrane pores. Binding is mediated by a C-terminally located tryptophan-rich motif. In a previous study, conformational effects of membrane binding were characterized by ... More
Fluorescent labeling of cysteine 39 on Escherichia coli primase places the dye near an active site.
AuthorsGriep MA, Mesman TN
JournalBioconjug Chem
PubMed ID8608179
'Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine. Its high chemical reactivity is likely due to its proximity to primase''s zinc, which is probably ligated to the adjacent residues 40-62. The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive. Primase ... More
Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability.
AuthorsKnight JL, Mekler V, Mukhopadhyay J, Ebright RH, Levy RM
JournalBiophys J
PubMed ID15542547
'We are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (FRET) measurements. In this article, we consider the problem of docking small-molecule ligands within macromolecular complexes. Using simulated FRET data, we ... More
Environment-sensitive labels in multiplex fluorescence analyses of protein-DNA complexes.
AuthorsDrees BL, Rye HS, Glazer AN, Nelson HC
JournalJ Biol Chem
PubMed ID8943271
'Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in ... More
Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides.
AuthorsNiesner U, Halin C, Lozzi L, Günthert M, Neri P, Wunderli-Allenspach H, Zardi L, Neri D
JournalBioconjug Chem
PubMed ID12121127
'Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution ... More
Soluble amyloid Abeta-(1-40) exists as a stable dimer at low concentrations.
AuthorsGarzon-Rodriguez W, Sepulveda-Becerra M, Milton S, Glabe CG
JournalJ Biol Chem
PubMed ID9261105
'Recent studies have implicated the amyloid Abeta peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble Abeta or its oligomeric state, and the existing data are often contradictory. In this study, we used intrinsic fluorescence ... More
Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer.
AuthorsNomanbhoy TK, Erickson JW, Cerione RA
JournalBiochemistry
PubMed ID10026253
'The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) to elicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In this report, we have set out to address this issue by using fluorescence resonance energy transfer approaches to examine the functional interactions of the RhoGDI ... More
Calcium and hydrogen ion concentrations in the parasitophorous vacuoles of epithelial cells infected with the microsporidian Encephalitozoon hellem.
AuthorsLeitch GJ, Scanlon M, Visvesvara GS, Wallace S
JournalJ Eukaryot Microbiol
PubMed ID7581320
'Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous ... More
Characterization of the uptake and toxicity of a fluorescent thiol reagent.
AuthorsOlive PL, Biaglow JE, Varnes ME, Durand RE
JournalCytometry
PubMed ID6839884
'3-(4-Maleimidylphenyl)-4-methyl-7-diethylamino coumarin (CPM), is a fluorescent thiol-binding agent. CPM is nontoxic to Chinese hamster V-79 cells (2 x 10(5) cells/ milliliter) exposed to 2.5 micrograms/milliliter for 30 minutes. However, both toxicity and cellular binding were directly dependent on the drug:cell ratio. Using flow cytometry, cellular binding of CPM correlated with ... More
Membrane-binding peptide from the C2 domain of factor VIII forms an amphipathic structure as determined by NMR spectroscopy.
AuthorsGilbert GE, Baleja JD
JournalBiochemistry
PubMed ID7893714
'Factor VIII binds to cell membranes prior to assembling with the serine protease, factor IXa, to form the factor X-activating enzyme complex. In order to better understand the interaction between factor VIII and phosphatidylserine-containing membranes, we have synthesized the membrane-binding peptide from the C2 domain of factor VIII, corresponding to ... More
Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents.
AuthorsFeng Y, Forgac M
JournalJ Biol Chem
PubMed ID1532573
'The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine ... More
The membrane topology of proton-pumping Escherichia coli transhydrogenase determined by cysteine labeling.
AuthorsMeuller J, Rydström J
JournalJ Biol Chem
PubMed ID10383409
'The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4''-maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and ... More
The distance between S1, S21, and the 3' end of 16S RNA in 30S ribosomal subunits. The effect of poly(uridylic acid) and 50S subunits on these distances.
AuthorsOdom OW, Dabbs ER, Dionne C, Müller M, Hardesty B
JournalEur J Biochem
PubMed ID6378636
'The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3'' end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non-radiative energy transfer to be: S21-16S RNA, 5.1 nm; S21-S1, 6.9 nm; S1-16S RNA, 6.8 nm. Binding of poly(uridylic ... More
Isolation and characterization of thrombin-activated human factor VIII.
AuthorsCurtis JE, Helgerson SL, Parker ET, Lollar P
JournalJ Biol Chem
PubMed ID8119969
'Recombinant human factor VIII (fVIII) was activated by thrombin at pH 7.4, followed by CM-Sepharose chromatography at pH values ranging from 3.5 to 7.4. Optimal coagulant activity was recovered at pH 5.5 and was associated with the isolation of an A1/A2/A3-C1-C2 heterotrimer. The activity was stable at -80 degrees C, ... More
SecA supports a constant rate of preprotein translocation.
AuthorsTomkiewicz D, Nouwen N, van Leeuwen R, Tans S, Driessen AJ
JournalJ Biol Chem
PubMed ID16601117
'In Escherichia coli, secretory proteins (preproteins) are translocated across the cytoplasmic membrane by the Sec system composed of a protein-conducting channel, SecYEG, and an ATP-dependent motor protein, SecA. After binding of the preprotein to SecYEG-bound SecA, cycles of ATP binding and hydrolysis by SecA are thought to drive the stepwise ... 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
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
Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor.
AuthorsRye HS, Drees BL, Nelson HC, Glazer AN
JournalJ Biol Chem
PubMed ID8227088
'The gel mobility-shift assay is an important tool for the study of protein-nucleic acid interactions. High detection sensitivity is typically attained by radioisotopic labeling of the target nucleic acid fragments. A novel fluorescence methodology offers significant advantages over this conventional approach. Ethidium, thiazole orange, and oxazole yellow homodimers form stable, ... 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
Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel.
AuthorsGlauner KS, Mannuzzu LM, Gandhi CS, Isacoff EY
JournalNature
PubMed ID10617202
'Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing ... More
Monitoring the cellular surface display of recombinant proteins by cysteine labeling and flow cytometry.
AuthorsJose J, Handel S
JournalChembiochem
PubMed ID12740811
'A general method is described that allows one to follow the surface display of recombinant proteins in Escherichia coli without having to use specific antibodies or enzymatic reactions. The method is based on cysteine-specific labeling through Michael addition to the double bond of maleimide and its derivatives, and takes advantage ... More
Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.
AuthorsRao A, Martin P, Reithmeier RA, Cantley LC
JournalBiochemistry
PubMed ID497152
'The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4''-isothiocyanostilbene-2,2''-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4''-dibenzamidostilbene-2,2''-disulfonate (DBDS) and 4-benzamido-4''-aminostilbene-2,2''-disulfonate (BADS) show a ... More
Topological analysis of the peripheral benzodiazepine receptor in yeast mitochondrial membranes supports a five-transmembrane structure.
AuthorsJoseph-Liauzun E, Delmas P, Shire D, Ferrara P
JournalJ Biol Chem
PubMed ID9442055
'The peripheral benzodiazepine receptor, implicated in the transport of cholesterol from the outer to the inner mitochondrial membrane, is predicted by hydropathy analysis to feature five membrane-spanning domains, with the amino terminus within the mitochondrial periplasm and the carboxyl terminus in the external cytoplasm. We have tested these structural predictions ... More
Conformations of IgE bound to its receptor Fc epsilon RI and in solution.
AuthorsZheng Y, Shopes B, Holowka D, Baird B
JournalBiochemistry
PubMed ID1832555
'Previous resonance energy transfer studies suggested that murine immunoglobulin E (IgE) is bent near the junction of its Fc and Fab segments when bound to its high-affinity receptor (Fc epsilon RI) on RBL cells. To examine further the conformations of IgE, both bound to this receptor and in solution, a ... More
Dissociation of the DNA polymerase III holoenzyme beta 2 subunits is accompanied by conformational change at distal cysteines 333.
AuthorsGriep MA, McHenry CS
JournalJ Biol Chem
PubMed ID2243096
'The beta subunit of DNA polymerase III holoenzyme is in a dimer-monomer equilibrium at physiological beta concentrations. Dissociation is accompanied by the fluorescence enhancement of a fluorophore attached to a unique sulfhydryl group of beta (Griep, M. A., and McHenry, C. S. (1988) Biochemistry 27, 5210-5215). Sequencing of the isolated ... More
The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein.
AuthorsRothnie A, Storm J, Campbell J, Linton KJ, Kerr ID, Callaghan R
JournalJ Biol Chem
PubMed ID15192095
'Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of ... More
D38 is an essential part of the proton translocation pathway in bacteriorhodopsin.
'At present, almost no knowledge exists about the functional relevance of the amino acid residues at the cytoplasmic (CP) surface of the light-driven proton pump bacteriorhodopsin (BR) although a prerequisite for efficient vectorial proton translocation is the efficient capture of protons from the alkaline cytoplasm of the cell. To identify ... More
Location of conserved residue histidine-38 of the epsilon subunit of Escherichia coli ATP synthase.
AuthorsSkakoon EN, Dunn SD
JournalArch Biochem Biophys
PubMed ID7682392
'The function and location of residue His-38 of the epsilon subunit of the Escherichia coli F1-ATPase were investigated. His-38 was replaced by glutamine and cysteine through site-directed mutagenesis to produce epsilon H38Q and epsilon H38C, respectively. Both epsilon H38Q and epsilon H38C fulfilled epsilon function in vivo as determined by ... More
DnaK-sigma 32 interaction is temperature-dependent. Implication for the mechanism of heat shock response.
AuthorsChattopadhyay R, Roy S
JournalJ Biol Chem
PubMed ID12084715
'The heat shock response in bacteria is a complex phenomenon in which sigma 32 plays the central role. The DnaK/J chaperone system binds and promotes degradation of sigma 32 at lower temperatures. At heat shock temperatures, the DnaK/J-mediated degradation of sigma 32 is largely abolished by a mechanism, which is ... More
Resonance energy transfer between sites in rat liver glutathione S-transferase, 1-1, selectively modified at cysteine-17 and cysteine-111.
AuthorsHu L, Colman RF
JournalBiochemistry
PubMed ID9048547
'Monobromobimane (mBBr) can label both Cys111 and Cys17 of rat liver glutathione S-transferase, 1-1 (GST 1-1). However, selective modification of Cys111 was achieved by the maleimide-based sulfhydryl reagents N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubation of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 ... More
The human T-cell leukemia virus-1 transcriptional activator Tax enhances cAMP-responsive element-binding protein (CREB) binding activity through interactions with the DNA minor groove.
'Tax-1, the transcriptional activation protein of human T-cell leukemia virus-1, increases transcription from the human T-cell leukemia virus-1 long terminal repeat and specific cellular promoters through interactions with cellular DNA-binding proteins. The Tax response elements (TxREs) of the long terminal repeat resemble cAMP response elements (CREs), the target of cAMP-responsive ... More
Inhibition of vacuolar H(+)-ATPase by disulfide bond formation between cysteine 254 and cysteine 532 in subunit A.
AuthorsFeng Y, Forgac M
JournalJ Biol Chem
PubMed ID8175752
'We have previously demonstrated that the coated vesicle vacuolar H(+)-ATPase (V-ATPase) can be inactivated by formation of intramolecular disulfide bonds (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 19769-19772). The disulfide bond responsible for inactivation can be distinguished from other disulfide bonds that form by the fact that ... More
Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation.
AuthorsMehan RS, White NC, Falke JJ
JournalBiochemistry
PubMed ID12627961
'The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and ... More
A competitive fluorescence assay to measure the reactivity of compounds.
AuthorsEpps DE, Taylor BM
JournalAnal Biochem
PubMed ID11476550
'A sensitive competitive method was developed for assessing the reactivity of compounds toward glutathione and toward thiols in general. The method employs the reaction of the fluorogenic reagent fluorescein-5-maleimide (FM) with glutathione (GSH) to generate a large increase in fluorescence emission. When the reaction is measured in the presence of ... More
Identification of a region of the H,K-ATPase alpha subunit associated with the beta subunit.
AuthorsShin JM, Sachs G
JournalJ Biol Chem
PubMed ID8132592
'The gastric H,K-ATPase consists of an alpha, beta heterodimer. To determine which part of the alpha subunit is associated with the beta subunit, hog gastric vesicles were trypsinized, which left most of the beta subunit undigested. The vesicles were solubilized with either C12E8 (polyoxyethylene 8 lauryl ester) or Nonidet P-40 ... More
Activating amphiphiles cause a conformational change of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes according to proteolytic digestion.
AuthorsLi L, Karlsson OP, Wieslander A
JournalJ Biol Chem
PubMed ID9368025
'1,2-Diacylglycerol 3-glucosyltransferase synthesizes the major nonbilayer-prone lipid monoglucosyldiacylglycerol (MGlcDAG) in the membrane of Acholeplasma laidlawii, which is important for the spontaneous curvature, and is a regulatory site for the lipid surface charge density. A potential connection between activity and a conformational change of this enzyme, governed by essential lipid activators, ... More
Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].
AuthorsGuevara J, Spurlino J, Jan AY, Yang CY, Tulinsky A, Prasad BV, Gaubatz JW, Morrisett JD
JournalBiophys J
PubMed ID8386013
'The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue ... More
Dimerization of the gastric H+, K(+)-ATPase.
AuthorsShin JM, Sachs G
JournalJ Biol Chem
PubMed ID8567637
'When the gastric H+, K(+)-ATPase was solubilized by n-dodecyl beta-D-maltoside and electrophoresed in blue native-polyacrylamide gels (BN-PAGE), one major band at about 360 kDa was observed. Since this band was recognized by both monoclonal antibodies 1218 (anti-alpha) and wheat germ agglutinin (anti-beta), the H+, K(+)-ATPase in its native state exists ... 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
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Intramolecular distances within the Ca(2+)-ATPase from sarcoplasmic reticulum as estimated through fluorescence energy transfer between probes.
'Fluorescence energy transfer measurements have been carried out to estimate intramolecular distances between probes bound to Ca(2+)-transporting ATPase (Ca(2+)-ATPase) as well as distances between these probes and the phospholipid headgroup. The nucleotide binding site was monitored by using 1,N6-ethenoadenosine 5''-triphosphate, a fluorescent analogue of ATP, and also by labelling Lys515 ... More
Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling.
'Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the ... More
Assembly, disassembly, and exchange of glial fibrillary acidic protein.
AuthorsNakamura Y, Takeda M, Angelides KJ, Tada K, Hariguchi S, Nishimura T
JournalGlia
PubMed ID1828780
'The kinetics and dynamics of glial fibrillary acidic protein (GFAP) assembly was explored by a fluorescence energy transfer assay method. Purified GFAP was stoichiometrically labeled at a single cysteine residue with fluorescein-maleimide. Soluble labeled GFAP in a low ionic strength buffer was assembled into 10 nm filaments by rapidly increasing ... More
Membrane insertion of the heptameric staphylococcal alpha-toxin pore. A domino-like structural transition that is allosterically modulated by the target cell membrane.
AuthorsValeva A, Schnabel R, Walev I, Boukhallouk F, Bhakdi S, Palmer M
JournalJ Biol Chem
PubMed ID11279048
'Staphylococcal alpha-toxin forms heptameric pores on eukaryotic cells. After binding to the cell membrane in its monomeric form, the toxin first assembles into a heptameric pre-pore. Subsequently, the pre-pore transforms into the final pore by membrane insertion of an amphipathic beta-barrel, which comprises the "central loop" domains of all heptamer ... More
Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase.
AuthorsTang C, Capaldi RA
JournalJ Biol Chem
PubMed ID8621695
'The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The ... More
Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.
AuthorsDuffy EJ, Parker ET, Mutucumarana VP, Johnson AE, Lollar P
JournalJ Biol Chem
PubMed ID1512239
'The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was ... More
Subunit structure and function of porcine factor Xa-activated factor VIII.
AuthorsParker ET, Pohl J, Blackburn MN, Lollar P
JournalBiochemistry
PubMed ID9235979
'Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report ... More
Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding.
AuthorsThevenin BJ, Periasamy N, Shohet SB, Verkman AS
JournalProc Natl Acad Sci U S A
PubMed ID8127875
'Interactions between the erythrocyte membrane and its skeleton are mediated primarily by binding of cytoskeletal components to a conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To examine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hinge ... More
Site-specific protein modification to identify the MutL interface of MutH.
AuthorsToedt GH, Krishnan R, Friedhoff P
JournalNucleic Acids Res
PubMed ID12560476
'We have mapped the region for the protein interaction site of the Escherichia coli mismatch repair protein MutH for its activator protein MutL by a site-specific protein modification approach. For this purpose we generated a cysteine-free variant of MutH and 12 variants thereof, each containing a single cysteine residue at ... More
Ca2+-dependent binding of severin to actin: a one-to-one complex is formed.
AuthorsGiffard RG, Weeds AG, Spudich JA
JournalJ Cell Biol
PubMed ID6427234
'Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. ... More
Actin filaments undergo limited subunit exchange in physiological salt conditions.
AuthorsPardee JD, Simpson PA, Stryer L, Spudich JA
JournalJ Cell Biol
PubMed ID7202009
'The exchange of actin filament subunits for unpolymerized actin or for subunits in other filaments has been quantitated by three experimental techniques: fluorescence energy transfer, incorporation of 35S-labeled actin monomers into unlabeled actin filaments, and exchange of [14C]ATP with filament-bound ADP. In the fluorescence energy transfer experiments, actin labeled with ... More
Biochemical identification of transmembrane segments of the Ca(2+)-ATPase of sarcoplasmic reticulum.
AuthorsShin JM, Kajimura M, Argüello JM, Kaplan JH, Sachs G
JournalJ Biol Chem
PubMed ID8077201
'The transmembrane segments of sarcoplasmic reticulum Ca(2+)-ATPase were determined by trypsinization of cytoplasmic side-out intact sarcoplasmic reticulum vesicles. The membrane portion of tryptic digest comprising the transmembrane fragments, joined by the intravesicular segments, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after labeling with fluorescein 5-maleimide in the presence of ... More
Identification of a region of the herpes simplex virus single-stranded DNA-binding protein involved in cooperative binding.
AuthorsDudas KC, Ruyechan WT
JournalJ Virol
PubMed ID9420222
'We have identified a region of the herpes simplex virus major DNA-binding protein (ICP8) which is involved in cooperative binding to single-stranded DNA. This has been accomplished by analysis of ICP8 which was covalently modified by reaction with the extrinsic fluorophore fluorescein-5-maleimide (FM). Reaction conditions which result in the incorporation ... More
Conformational change in the herpes simplex single-strand binding protein induced by DNA.
AuthorsDudas KC, Scouten SK, Ruyechan WT
JournalBiochem Biophys Res Commun
PubMed ID11594771
'Protease digestion of the herpes simplex virus type 1 major single-strand DNA binding protein ICP8 showed that the cleavage patterns observed in the presence and absence of single-stranded DNA oligonucleotides are substantially different with protection of cleavage sites between amino acids 293 and 806 observed in the presence of oligonucleotide. ... More
Binding of bovine factor Va to phosphatidylcholine membranes.
AuthorsKoppaka V, Lentz BR
JournalBiophys J
PubMed ID8744331
'The interaction of bovine factor Va with phosphatidylcholine membranes was examined using four different fluorescence techniques: 1) changes in the fluorescence anisotropy of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to monitor the interaction of factor Va with 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), 2) changes in the fluorescence anisotropy of ... More
Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes.
AuthorsThomas DD, Stryer L
JournalJ Mol Biol
PubMed ID7077659
Use of fluorescence spectroscopy to study conformational changes in the beta 2-adrenoceptor.
AuthorsKobilka BK, Gether U
JournalMethods Enzymol
PubMed ID11665566
Simple blocking system for use with maleimide-labeled nucleic acid probes.
AuthorsFung J, Moronne MM, Weier HU
JournalBiotechniques
PubMed ID9762435
Structure-function relationship in Escherichia coli initiation factors. Environment of the Cys residue and evidence for a hydrophobic region in initiation factor IF3 by fluorescence and ESR spectroscopy.
AuthorsPon C, Cannistraro S, Giovane A, Gualerzi C
JournalArch Biochem Biophys
PubMed ID6289754
Fluorescent localization of contractile proteins in tissue culture cells.
AuthorsWang K, Feramisco JR, Ash JF
JournalMethods Enzymol
PubMed ID6750319
Exposure of actin thiols by the removal of tightly held calcium ions.
AuthorsKonno K, Morales MF
JournalProc Natl Acad Sci U S A
PubMed ID3865205
The removal of bound metal ions from G-actin uncovered two thiols, Cys-10 and Cys-257. The uncovering of these thiols requires a free calcium concentration lower than 10 nM. Therefore, participation of one or both thiols in Ca2+ binding is suggested. Actin labeled with N-(5-fluoresceinyl)maleimide in the absence of calcium moves ... More
Demonstration of sulfhydryl and disulfide groups by a fluorescent maleimide procedure.
AuthorsCurtis SK, Cowden RR
JournalHistochemistry
PubMed ID7190963
Several fluorescent maleimide compounds were evaluated as possible substitutes for N-(4-aminophenyl)maleimide in the histochemical procedures developed by Sippel (1973, 1978a, b, 1980) for the demonstration of sulfhydryl and disulfide groups. The brightest and most selective fluorescence was obtained by using N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), although both eosin-5-maleimide and fluorescein-5-maleimide could also be ... More
The core of the bacterial translocase harbors a tilted transmembrane segment 3 of SecE.
AuthorsVeenendaal AK, Van Der Does C, Driessen AJ
JournalJ Biol Chem
PubMed ID12138117
The bacterial translocase mediates the translocation and membrane integration of proteins. The integral membrane proteins SecY and SecE are conserved core subunits of the translocase. Previous cysteine-scanning studies showed that the transmembrane segment (TMS) 3 of SecE contacts TMS 2 and 7 of SecY, and TMS 3 of another SecE. ... More
Distribution of protein sulphydryls and disulphides in fixed mammalian chromosomes, and their relationship to banding.
AuthorsSumner AT,
JournalJ Cell Sci
PubMed ID6501434
Fixed mammalian mitotic chromosomes, when stained with a variety of sensitive, sulphydryl-specific fluorochromes directly or following reduction of disulphide groups, show uniform fluorescence. Thus sulphydryl and disulphide groups are uniformly distributed along the length of the chromosomes, and do not show patterns related to chromosome bands. Performance of G- or ... More
Site-specific fluorescence labelling of recombinant polyomavirus-like particles.
AuthorsSchmidt U, Kenklies J, Rudolph R, Böhm G
JournalBiol Chem
PubMed ID10223344
For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery. Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas ... More
Enhanced binding of altered H-NS protein to flagellar rotor protein FliG causes increased flagellar rotational speed and hypermotility in Escherichia coli.
AuthorsDonato GM, Kawula TH
JournalJ Biol Chem
PubMed ID9727020
H-NS is an Escherichia coli nucleoid protein known only to function as a modulator of gene expression. In this study, we found that specific single amino acid substitutions in H-NS caused an approximately 50% increase in flagellum rotational speed. In fluorescence anisotropy and chemical cross-linking assays, H-NS interacted with the ... More
Structure of a bacterial sensory receptor. A site-directed sulfhydryl study.
AuthorsFalke JJ, Dernburg AF, Sternberg DA, Zalkin N, Milligan DL, Koshland DE
JournalJ Biol Chem
PubMed ID3049592
Cysteines are substituted at six positions in the aspartate receptor, and these mutant proteins are used to investigate three major facets of receptor structure. 1) The surface of the receptor is examined through measurement of the rate constants for chemical modification of the cysteines by aqueous reagents. Different positions exhibit ... More
Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other ... More
The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain.
AuthorsYou M, Li E, Hristova K
JournalBiochemistry
PubMed ID16634636
The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to ... More
Cysteine-specific radioiodination of proteins with fluorescein maleimide.
AuthorsPalmer M, Buchkremer M, Valeva A, Bhakdi S
JournalAnal Biochem
PubMed ID9367500
A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label ... More
Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes.
AuthorsOdom OW, Picking WD, Hardesty B
JournalBiochemistry
PubMed ID1703007
The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more ... 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
Antiparallel dimer and actin assembly.
AuthorsGrintsevich EE, Phillips M, Pavlov D, Phan M, Reisler E, Muhlrad A,
JournalBiochemistry
PubMed ID20361759
The antiparallel dimer (APD) is a unique actin species, which can be detected in the early stages of actin polymerization. In this work, we introduce novel tools for examination of the effects of the APD on actin polymerization. We document that bifunctional methanothiosulfonate (MTS) reagents are an attractive alternative to ... More
Fluorescence applications in molecular neurobiology.
AuthorsTaraska JW, Zagotta WN,
JournalNeuron
PubMed ID20434995
Macromolecules drive the complex behavior of neurons. For example, channels and transporters control the movements of ions across membranes, SNAREs direct the fusion of vesicles at the synapse, and motors move cargo throughout the cell. Understanding the structure, assembly, and conformational movements of these and other neuronal proteins is essential ... More
Proton migration along the membrane surface and retarded surface to bulk transfer.
AuthorsHeberle J, Riesle J, Thiedemann G, Oesterhelt D, Dencher NA
JournalNature
PubMed ID8047144
Since the proposal of the chemiosmotic theory there has been a continuing debate about how protons that have been pumped across membranes reach another membrane protein that utilizes the established pH gradient. Evidence has been gathered in favour of a 'delocalized' theory, in which the pumped protons equilibrate with the ... More
Use of luminescence resonance energy transfer to measure distances in the AE1 anion exchange protein dimer.
AuthorsKnauf PA, Pal P
JournalBlood Cells Mol Dis
PubMed ID15121092
To understand how red blood cell and other proteins carry out their functions, it is necessary not only to have high-resolution crystal structures, but also to have methods that can measure changes in position of parts of the protein on the scale of Angstroms. The method of luminescence resonance energy ... More
Conformational changes involved in thermal aggregation processes of bovine serum albumin.
AuthorsMilitello V, Vetri V, Leone M
JournalBiophys Chem
PubMed ID12932585
We report a kinetic study on thermal aggregation process of the model protein bovine serum albumin (BSA) in low concentration regime. Aim of this study is to provide information on relationship between conformational changes and initial step of aggregation. The experimental approach is based on steady-state fluorescence spectra of the ... More
HLA-DM targets the hydrogen bond between the histidine at position beta81 and peptide to dissociate HLA-DR-peptide complexes.
AuthorsNarayan K, Chou CL, Kim A, Hartman IZ, Dalai S, Khoruzhenko S, Sadegh-Nasseri S
JournalNat Immunol
PubMed ID17143275
The peptide editor HLA-DM (DM) mediates exchange of peptides bound to major histocompatibility (MHC) class II molecules during antigen processing; however, the mechanism by which DM displaces peptides remains unclear. Here we generated a soluble mutant HLA-DR1 with a histidine-to-asparagine substitution at position 81 of the beta-chain (DR1betaH81N) to perturb ... More
A robust method for production of MHC tetramers with small molecule fluorophores.
AuthorsRamachandiran V, Grigoriev V, Lan L, Ravkov E, Mertens SA, Altman JD
JournalJ Immunol Methods
PubMed ID17187819
Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While ... More
Reversible dimer dissociation of tubulin S and tubulin detected by fluorescence anisotropy.
AuthorsPanda D, Roy S, Bhattacharyya B
JournalBiochemistry
PubMed ID1390747
Concentration-dependent dissociation of dimers of goat brain tubulin S and tubulin was studied by fluorescence anisotropy. Upon dilution, assembly-competent fluorescein 5'-maleimide labeled dimers of tubulin S and tubulin show a progressive decrease in fluorescence anisotropy. That this lowering of anisotropy results from the dissociation of tubulin S dimers into monomers ... More
Real-time observation of trigger factor function on translating ribosomes.
AuthorsKaiser CM, Chang HC, Agashe VR, Lakshmipathy SK, Etchells SA, Hayer-Hartl M, Hartl FU, Barral JM
JournalNature
PubMed ID17051157
The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating ... More
Localization of the elongation factor Tu binding site on Escherichia coli ribosomes.
AuthorsRychlik W, Odom OW, Hardesty B
JournalBiochemistry
PubMed ID6338919
Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a ... More
Avidin-FITC topological studies with three cysteine mutants of equinatoxin II, a sea anemone pore-forming protein.
AuthorsAnderluh G, Barlic A, Krizaj I, Menestrina G, Gubensek F, Macek P
JournalBiochem Biophys Res Commun
PubMed ID9439633
Equinatoxin II (EqtII) is a cysteinless pore-forming protein from sea anemone Actinia equina. Three cysteine mutants were produced in an E. coli expression system in order to study the topology of lysine 77, arginine 126, and alanine 179. Accessibility of an introduced thiol group in the water soluble mutants was ... More
Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor.
AuthorsGhanouni P, Gryczynski Z, Steenhuis JJ, Lee TW, Farrens DL, Lakowicz JR, Kobilka BK
JournalJ Biol Chem
PubMed ID11320077
G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain ... More
SDS capillary gel electrophoresis of proteins in microfabricated channels.
AuthorsYao S, Anex DS, Caldwell WB, Arnold DW, Smith KB, Schultz PG
JournalProc Natl Acad Sci U S A
PubMed ID10318890
Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations ... More
Fluorescence resonance energy transfer mapping of subunit delta in spinach chloroplast F1 ATPase.
AuthorsEngelbrecht S, Giakas E, Marx O, Lill H
JournalEur J Biochem
PubMed ID9523699
Despite the considerable progress in the field of F0F1-ATPases caused by solving the 2.8-A structure of mitochondrial F1 ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628], little is known about the position and function of the enzyme's small subunits which ... More
Phosphatidylethanolamine induces high affinity binding sites for factor VIII on membranes containing phosphatidyl-L-serine.
AuthorsGilbert GE, Arena AA
JournalJ Biol Chem
PubMed ID7629178
Synthetic membranes of phosphatidylcholine require inclusion of at least 5% phosphatidylserine (Ptd-L-Ser) to form binding sites for factor VIII. The relatively high requirement for Ptd-L-Ser suggests that stimulated platelets may contain another membrane constituent that enhances expression of factor VIII-binding sites. We report that phosphatidylethanolamine (PE), which is exposed in ... More
Voltage-clamp fluorometry in the local environment of the C255-C511 disulfide bridge of the Na+/glucose cotransporter.
AuthorsGagnon DG, Frindel C, Lapointe JY
JournalBiophys J
PubMed ID17208964
We recently identified a functionally important disulfide bridge between C255 and C511 of the human Na+/glucose cotransporter SGLT1. In this study, voltage-clamp fluorometry was used to characterize the fluorescence of four different dyes attached to C255 and C511 under various ionic and substrate/inhibitor conditions. State-dependent fluorescence changes (DeltaF) were observed ... More
The dimer of the beta subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II).
AuthorsGriep MA, McHenry CS
JournalBiochemistry
PubMed ID3048397
The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding ... More
Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles.
AuthorsDe Keyzer J, Van Der Does C, Driessen AJ
JournalJ Biol Chem
PubMed ID12226104
Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, ... More
Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor.
AuthorsGhanouni P, Steenhuis JJ, Farrens DL, Kobilka BK
JournalProc Natl Acad Sci U S A
PubMed ID11353823
The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) ... More