Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase.
AuthorsBhattacharjee H,Rosen BP
JournalThe Journal of biological chemistry
PubMed ID8798705
The soft metal ion binding sites in the Staphylococcus aureus pI258 CadC Cd(II)/Pb(II)/Zn(II)-responsive repressor are formed between subunits of the homodimer.
Authors Wong Marco D; Lin Yung-Feng; Rosen Barry P;
JournalJ Biol Chem
PubMed ID12176999
'The Staphylococcus aureus plasmid pI258 CadC is a homodimeric repressor that binds Cd(II), Pb(II), and Zn(II) and regulates expression of the cadAC operon. CadC binds two Cd(II) ions per dimer, with a tetrathiolate binding site composed of residues Cys(7), Cys(11), Cys(58), and Cys(60). It is not known whether each site ... More
Dibromobimane as a fluorescent crosslinking reagent.
AuthorsKim JS, Raines RT
JournalAnal Biochem
PubMed ID7778775
Modification of the actin interface of skeletal myosin subfragment-1 by treatment with dibromobimane.
AuthorsMornet D, Ue K, Chaussepied P, Morales MF
JournalEur J Biochem
PubMed ID3758077
'Recently, by treating the head portion of skeletal myosin subfragment-1 (S1) with the bifunctional agent dibromobimane, we introduced an intramolecular covalent cross-link which resulted in the stabilisation of an internal loop in the heavy chain structure of the head [Mornet et al. (1984) Proc. Natl Acad. Sci. USA 82, 1658-1662]. ... More
Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID12223492
'The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 ... More
Identification of the 29,000-dalton protein and its relevance to oligomycin-sensitive 32Pi-ATP exchange in bovine heart electron transport particles.
AuthorsJoshi S, Torok K
JournalJ Biol Chem
PubMed ID6238028
'There have been several reports on the involvement of a 29,000-dalton protein in the regulation of ATP synthesis and 32Pi-ATP exchange (Zimmer, G., Mainka, L., and Heil, B. M. (1982) FEBS Lett. 150, 207-210). The present communication demonstrates that incubation of electron transport particles with 50 microM copper-o-phenanthroline results in ... More
Transmembrane domain (TM) 9 represents a novel site in P-glycoprotein that affects drug resistance and cooperates with TM6 to mediate [125I]iodoarylazidoprazosin labeling.
AuthorsSong J, Melera PW
JournalMol Pharmacol
PubMed ID11455011
'The multidrug resistant cell line DC-3F/ADII was obtained by stepwise selection for growth in actinomycin D (ActD). Compared with parental cells, it displays high resistance to ActD and vincristine and low resistance to colchicine and daunorubicin. These cells overexpress a form of P-glycoprotein (Pgp1) containing a double mutation, I837L and ... More
Temperature-induced changes in the flexibility of the loop between SH1 (Cys-707) and SH3 (Cys-522) in myosin subfragment 1 detected by cross-linking.
AuthorsAgarwal R, Burke M
JournalArch Biochem Biophys
PubMed ID1898079
'The ability of dibromobimane to cross-link SH1 (Cys-707) in the 21-kDa C-terminal segment to SH3 (Cys-522) in the 50-kDa middle segment of the myosin S1 heavy chain has been examined as a function of nucleotide binding and temperature. The results obtained indicate that, while the reagent rapidly reacts with SH1 ... More
Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain.
AuthorsChen Z, Akin BL, Stokes DL, Jones LR
JournalJ Biol Chem
PubMed ID16554295
'Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+-phenanthroline, dibromobimane, and ... More
Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID11279063
'Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of ... More
Bimane fluorescent labels. Characterization of the bimane labeling of human hemoglobin.
AuthorsKosower NS, Newton GL, Kosower EM, Ranney HM
JournalBiochim Biophys Acta
PubMed ID7378449
'The products of the bimane labeling (using a monobromobimane, a dibromobimane and a quaternary bromobimane) of hemoglobin are characterized. Peptide mapping identifies cysteine-beta 93 as the reactive thiol site. Electrophoretic mobility of hemoglobin varies with the label used, that of monobromobimane-labeled hemoglobin being unaltered, while dibromobimane- and trimethylammoniobromobimane-labeled hemoglobin exhibit ... More
Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli.
AuthorsWu J, Voss J, Hubbell WL, Kaback HR
JournalProc Natl Acad Sci U S A
PubMed ID8816762
'Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and ... More
The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID10681495
'Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the ... More
Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID10585407
'The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase ... More
Proximity and ligand-induced movement of interdomain residues in myosin subfragment 1 containing trapped MgADP and MgPPi probed by multifunctional cross-linking.
AuthorsRajasekharan KN, Sivaramakrishnan M, Burke M
JournalJ Biol Chem
PubMed ID3038911
'The conformations of myosin subfragment 1 containing trapped MgADP or MgPPi have been studied by investigating the spatial disposition of the remainder of the subfragment 1 structure to the covalently bridged ATPase-related thiols SH1 and SH2. This has been done by synthesizing a trifunctional photoactivatable reagent 4,4''-bis(N-maleimido)benzophenone and reacting it ... More
Effect of sulfhydryl group modification on the activity of bovine ferrochelatase.
AuthorsDailey HA
JournalJ Biol Chem
PubMed ID6698990
'The role of sulfhydryl groups in the activity of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), has been examined by using a variety of sulfhydryl group-specific reagents. The enzyme is rapidly inactivated in a pseudo-first order reaction by N-ethylmaleimide and monobromobimane and more slowly ... More
Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID11013259
'P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1-5 ... More
Bromobimane probes for thiols.
AuthorsKosower EM, Kosower NS
JournalMethods Enzymol
PubMed ID7651193
Thiol labeling with bromobimanes.
AuthorsKosower NS, Kosower EM
JournalMethods Enzymol
PubMed ID3657564
Stabilization of a primary loop in myosin subfragment 1 with a fluorescent crosslinker.
AuthorsMornet D, Ue K, Morales MF
JournalProc Natl Acad Sci U S A
PubMed ID3856845
A bifunctional fluorescent alkylating agent, dibromobimane, has been used to stabilize a preexisting primary loop in myosin subfragment 1 (S-1). The crosslink achieved joins Cys-707 (called sulfhydryl group "SH1") of the 20-kDa domain (formerly called "20K" domain) with a thiol of the 50-kDa domain and seems to place the dibromobimane ... More
Quantitative evaluation of the lengths of homobifunctional protein cross-linking reagents used as molecular rulers.
AuthorsGreen NS, Reisler E, Houk KN
JournalProtein Sci
PubMed ID11420431
Homobifunctional chemical cross-linking reagents are important tools for functional and structural characterization of proteins. Accurate measures of the lengths of these molecules currently are not available, despite their widespread use. Stochastic dynamics calculations now provide quantitative measures of the lengths, and length dispersions, of 32 widely used molecular rulers. Significant ... More
Bromobimane crosslinking studies in oligomycin-sensitive ATPase from beef heart mitochondria. Mr 31 000 protein crosslinked.
AuthorsZimmer G, Mainka L, Heil BM
JournalFEBS Lett
PubMed ID6218999
Using a bromobimane fluorescent label the Mr 31 000 protein band oligomycin-sensitive (OS)-ATPase from beef heart mitochondria is shown to become much intensified by 2-mercaptopropionylglycine. In the presence of 3.5 nmol/mg protein of the thiol reagent ATP-Pi exchange activity is increased by 90%. With the fluorescent crosslinking reagent dibromobimane (DB) ... More
Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.
AuthorsKosower NS, Kosower EM, Newton GL, Ranney HM
JournalProc Natl Acad Sci U S A
PubMed ID291011
The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivatives of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently label hemoglobin (reactive thiol groups), membrane proteins, and glutathione of normal human red cells under physiological conditions. Monobromobimane and dibromobimane are effective on intact cells while red cell membranes may be impermeable to the positively charged ... More
Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain.
AuthorsTong SW, Elzinga M
JournalJ Biol Chem
PubMed ID2318873
The amino acid sequence of the 50-kDa fragment that is released by limited tryptic digestion of the head portion of rabbit skeletal muscle myosin was determined by analysis and alignment of sets of peptides generated by digestion of the fragment at arginine or methionine residues. This fragment contains residues 205-636 ... More
Intramolecular cross-linking of myosin subfragment 1 with bimane.
AuthorsUe K
JournalBiochemistry
PubMed ID3593699
We previously showed that the fluorescent inter-thiol cross-linker dibromobimane (DBB) [Kosower, N. S., Kosower, E. M., Newton, G. L., & Ranney, H. M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382-3386] cross-links two [50 and 20 kilodaltons (kDa)] of the three major fragments of myosin subfragment 1 (S-1); on intact ... More
The internal crosslinking of the S1 heavy chain from smooth muscle probed by dibromobimane.
AuthorsBonet A, Audemard E, Mornet D
JournalBiochem Biophys Res Commun
PubMed ID2965870
The reaction of the crosslinker dibromobimane has recently revealed a functionally important internal loop structure within the skeletal myosin S1 heavy chain where Cys-522 of the 50K domain and Cys-707 (SH1) of the 20K region are spatially juxtaposed. Here we have studied the possible relevance of this topological feature to ... More
Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET.
AuthorsBuskiewicz I, Peske F, Wieden HJ, Gryczynski I, Rodnina MV, Wintermeyer W,
JournalJ Mol Biol
PubMed ID16005894
The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure ... More
Consequences of placing an intramolecular crosslink in myosin S1.
AuthorsKonno K, Ue K, Khoroshev M, Martinez H, Ray B, Morales MF
JournalProc Natl Acad Sci U S A
PubMed ID10677484
This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, "S1," of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, ... More
Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork. Characterization of a fluorescently labeled DNA polymerase sliding clamp.
The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled ... More
Effect of cholesterol upon the conformation of band 3 and its transmembrane fragment.
AuthorsKlugerman AH, Gaarn A, Parkes JG
JournalCan J Biochem Cell Biol
PubMed ID6509361
Vesicles enriched in the anion transport protein band 3 and its transmembrane domain were prepared, and the cysteine residues were labelled with an extrinsic fluorescent probe, monobromobimane. Fluorescence energy transfer was demonstrated between intrinsic tryptophans and monobromobimane, and an average interchromophoric distance, Rav, was defined. Rav values and fluorescence emission ... More
Cross-linking of human multidrug resistance P-glycoprotein by the substrate, tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis. Evidence for rotation of a transmembrane helix.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID11429407
We identified a thiol-reactive substrate, Tris-(2-maleimidoethyl)amine (TMEA), to explore the contribution of the TM segments 6 and 12 of the human multidrug resistance P-glycoprotein (P-gp) during transport. TMEA is a trifunctional maleimide and stimulated the ATPase activity of Cys-less P-gp about 7-fold. Cysteine-scanning mutagenesis of TM12 showed that the activity ... More
Cross-linking constraints on F-actin structure.
AuthorsKim E, Wriggers W, Phillips M, Kokabi K, Rubenstein PA, Reisler E
JournalJ Mol Biol
PubMed ID10860749
The DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274), and the C terminus region are among the structural elements of monomeric (G-) actin proposed to form the intermonomer interface in F-actin. To test the proximity and interactions of these elements and to provide constraints on models of ... More
Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID11518701
The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4-6 and 10-12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) ... More
Mechanism of ribonuclease cytotoxicity.
AuthorsKim JS, Soucek J, Matousek J, Raines RT
JournalJ Biol Chem
PubMed ID8537370
Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pancreatic ribonuclease A (RNase A), is toxic to mammalian cells. In contrast to dimeric BS-RNase, a monomeric BS-RNase and RNase A are not cytotoxic and are bound tightly by cytosolic ribonuclease inhibitor. To elucidate the mechanism of ribonuclease cytotoxicity, we constructed ... More
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID9405384
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to ... More
Bromobimanes--fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections.
AuthorsDanielsohn P, Nolte A
JournalHistochemistry
PubMed ID3570877
Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: ... More
P-glycoprotein retains function when reconstituted into a sphingolipid- and cholesterol-rich environment.
AuthorsModok S, Heyward C, Callaghan R
JournalJ Lipid Res
PubMed ID15258203
P-glycoprotein (P-gp) appears to be associated within specialized raftlike membrane microdomains. The activity of P-gp is sensitive to its lipid environment, and a functional association in raft microdomains will require that P-gp retains activity in the microenvironment. Purified hamster P-gp was reconstituted in liposomes comprising sphingomyelin and cholesterol, both highly ... More
Identification and mapping of protein-protein interactions between gp32 and gp59 by cross-linking.
AuthorsIshmael FT, Alley SC, Benkovic SJ
JournalJ Biol Chem
PubMed ID11309384
The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA ... More
Mapping spatial proximities of sulfhydryl groups in proteins using a fluorogenic cross-linker and mass spectrometry.
AuthorsSinz A, Wang K
JournalAnal Biochem
PubMed ID15245993
Chemical cross-linking of proteins in combination with mass spectrometric analysis of the reaction products has gained renewed interest as a method of obtaining distance constraints within a protein and determining a low-resolution three-dimensional structure. We present a method for identifying spatially close sulfhydryl groups in proteins employing chemical cross-linking with ... More
Flow cytometry techniques for studying cellular thiols.
AuthorsDurand RE, Olive PL
JournalRadiat Res
PubMed ID6193555
Cellular thiols, and especially glutathione, act as scavenger nucleophiles and can protect against toxicity, mutagenicity, or transformation by ionizing radiation and many carcinogens. Development of a rapid assay to quantitate the cellular content of thiols could thus be useful in assessing or predicting cellular risk to damage. Several fluorescent thiol-reactive ... More
Plasmin reduction by phosphoglycerate kinase is a thiol-independent process.
AuthorsLay AJ, Jiang XM, Daly E, Sun L, Hogg PJ
JournalJ Biol Chem
PubMed ID11782484
Phosphoglycerate kinase (PGK) is secreted by tumor cells and facilitates reduction of disulfide bond(s) in plasmin (Lay, A. J., Jiang, X.-M., Kisker, O., Flynn, E., Underwood, A., Condron, R., and Hogg, P. J. (2000) Nature 408, 869-873). The angiogenesis inhibitor, angiostatin, is cleaved from the reduced plasmin by a combination ... More
Identification of a nonconserved amino acid residue in multidrug resistance protein 1 important for determining substrate specificity: evidence for functional interaction between transmembrane helices 14 and 17.
AuthorsZhang DW, Cole SP, Deeley RG
JournalJ Biol Chem
PubMed ID11429411
Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for ... More
Identification of ligand-binding regions of P-glycoprotein by activated-pharmacophore photoaffinity labeling and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.
AuthorsEcker GF, Csaszar E, Kopp S, Plagens B, Holzer W, Ernst W, Chiba P
JournalMol Pharmacol
PubMed ID11854445
Energy dependent efflux pumps confer resistance to anticancer, antimicrobial, and antiparasitic drugs. P-glycoprotein (Pgp, ABCB1) mediates resistance to a broad spectrum of antitumor drugs. Compounds that themselves are nontoxic to cells have been shown to act as inhibitors of Pgp. The mechanism of binding and transport of low-molecular-mass ligands by ... More