Fluorescence resonance energy transfer mapping of the fourth of six nucleotide-binding sites of chloroplast coupling factor 1.
AuthorsShapiro AB, Gibson KD, Scheraga HA, McCarty RE
JournalJ Biol Chem
PubMed ID1832671
Equilibrium dialysis measurements of adenine nucleotide binding to chloroplast coupling factor 1 suggest that the enzyme has six binding sites for ADP, adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), and 2'(3')-O-2,4,6-trinitrophenyl-ATP (TNP-ATP). High affinity binding at all six sites requires the divalent cation, Mg2+. Three of the nucleotide-binding sites, sites 1, 2, and 3, have ... 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
The pleckstrin homology domain of phospholipase Cbeta transmits enzymatic activation through modulation of the membrane-domain orientation.
AuthorsDrin G, Douguet D, Scarlata S
JournalBiochemistry
PubMed ID16669615
'Phospholipase Cbeta (PLCbeta) enzymes are activated by Galpha q and Gbetagamma subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Activation of PLCbeta2 by Gbetagamma subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain, although the underlying mechanism is unclear. Also ... 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
Signaling through a G Protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model.
AuthorsPhilip F, Sengupta P, Scarlata S
JournalJ Biol Chem
PubMed ID17420253
'The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the Galpha(q) family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the ... More
An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex.
AuthorsZhuang Z, Berdis AJ, Benkovic SJ
JournalBiochemistry
PubMed ID16800623
'In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form ... More
Regulation of the rate and extent of phospholipase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins.
AuthorsRunnels LW, Scarlata SF
JournalBiochemistry
PubMed ID9799521
'The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a ... 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
Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.
AuthorsHu D, Crist M, Duan X, Gimble FS
JournalBiochemistry
PubMed ID10504231
'The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that ... More
Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking.
AuthorsHu D, Crist M, Duan X, Quiocho FA, Gimble FS
JournalJ Biol Chem
PubMed ID10644733
'The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To ... 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
Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin.
AuthorsEssex DW, Miller A, Swiatkowska M, Feinman RD
JournalBiochemistry
PubMed ID10441134
'In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as ... More
Resonance energy transfer between the active sites of myocardial-type creatine kinase (isozyme MB).
AuthorsGrossman SH
JournalBiochemistry
PubMed ID6652070
'The single reactive sulfhydryl group, located in the active site of each subunit of dimeric creatine kinase from rabbit muscle (isozyme MM), was selectively labeled with 3-(4-maleimidylphenyl)-7-(diethylamino)-4-methylcoumarin (CPM). Isozyme BB, purified to homogeneity from rabbit brain, was conjugated with the sulfhydryl-specific reagent 5''-(iodoacetamido)fluorescein (5''-IAF). Spectral analyses demonstrated that 1.8 mol ... 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
Relaxation time, interthiol distance, and mechanism of action of ribosomal protein S1.
AuthorsOdom OW, Deng HY, Subramanian AR, Hardesty B
JournalArch Biochem Biophys
PubMed ID6201138
'The two sulfhydryl groups of ribosomal protein S1 from Escherichia coli have been labeled with fluorescent maleimides and the distance between them has been determined by nonradiative energy transfer. This distance was found to be approximately 27 A for both free S1 and S1 bound to 30 S subunits. This ... More
Structural implications of the chemical modification of Cys(10) on actin.
AuthorsEli-Berchoer L, Reisler E, Muhlrad A
JournalBiophys J
PubMed ID10692333
'Cys(10) is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys(10) was modified with fluorescence probes N-(iodoacetyl)N''-(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4''-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromo bimane (MBB) by the method of, J. Biol. Chem. 266:5508-5513). The specificity of Cys(10) ... More
Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.
AuthorsPicking WD, Odom OW, Hardesty B
JournalBiochemistry
PubMed ID1282033
'A coumarin derivative was covalently attached to either the amino acid or the 5'' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong ... More
Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome.
AuthorsRamachandiran V, Willms C, Kramer G, Hardesty B
JournalJ Biol Chem
PubMed ID10636875
'Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All ... More
Purification, cDNA cloning, and characterization of a new serpin with megakaryocyte maturation activity.
AuthorsTsujimoto M, Tsuruoka N, Ishida N, Kurihara T, Iwasa F, Yamashiro K, Rogi T, Kodama S, Katsuragi N, Adachi M, Katayama T, Nakao M, Yamaichi K, Hashino J, Haruyama M, Miura K, Nakanishi T, Nakazato H, Teramura M, Mizoguchi H, Yamaguchi N
JournalJ Biol Chem
PubMed ID9182567
'A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor ... More
The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: arginine 94 is important for the binding of OSCP to F1.
AuthorsGolden TR, Pedersen PL
JournalBiochemistry
PubMed ID9753477
'The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis. To gain insight into the function of OSCP, mutations were made in a highly conserved central region of ... More
Differential lymphocyte growth-modifying effects of oxidants: changes in cytosolic Ca+2.
AuthorsDuncan DD, Lawrence DA
JournalToxicol Appl Pharmacol
PubMed ID2506676
'An increase in the concentration of cytosolic Ca+2 ([Ca-2]i) is among the earliest changes seen in mitogen-stimulated lymphocytes and is a consequence of signal transduction which usually results in the initiation of cell cycle progression. However, increased [Ca+2]i has also been correlated with cytotoxicity. We have determined whether modulations of ... More
Measurement of distance between the active serine of the thioesterase domain and the pantetheine thiol of fatty acid synthase by fluorescence resonance energy transfer.
AuthorsFoster RJ, Poulose AJ, Bonsall RF, Kolattukudy PE
JournalJ Biol Chem
PubMed ID3919010
'Fatty acid synthase from the uropygial gland was inactivated by treatment with pyrenebutyl methanephosphonofluoridate by specific modification of the "active serine" at the thioesterase domain. Treatment of fatty acid synthase with 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin resulted in the loss of the condensation activity and overall synthase activity. Acetyl-CoA and malenyl-CoA protected the enzyme ... More
SV40 large T-antigen nuclear signal analogues: successful nuclear targeting with bovine serum albumin but not low molecular weight fluorescent conjugates.
AuthorsLobl TJ, Mitchell MA, Maggiora LL
JournalBiopolymers
PubMed ID2328286
'The signal sequence of a nuclear-directed protein encodes the necessary information for targeting the attached proteins to the cell nucleus. The sequence/structural requirements for a functional transport signal were explored with a series of peptides derived from the simian virus 40 large T-antigen nuclear signal 126-134 (CPKKKRKVED-NH2, wild type) conjugated ... 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
Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation.
AuthorsPicking WD, Picking WL, Odom OW, Hardesty B
JournalBiochemistry
PubMed ID1540593
'The fate of the amino termini of nascent polyalanine, polyserine, and polylysine was monitored by fluorescence techniques as each was translated on Escherichia coli ribosomes. A coumarin probe was placed at the alpha-amino group of a synthetic elongator alanyl-tRNA or a synthetic initiator alanyl-tRNA or at the epsilon-amino group of ... More
Determination of the affinities between heterotrimeric G protein subunits and their phospholipase C-beta effectors.
AuthorsRunnels LW, Scarlata SF
JournalBiochemistry
PubMed ID9931014
'Phosphatidylinositide-specific phospholipase C-betas play a key role in Ca2+ signaling and are specifically activated by the alphaq family of heterotrimeric G proteins and as well as betagamma subunits. We have determined the affinity between Gbetagamma subunits and GTPgammaS and GDP-liganded Galphaq subunits on membrane surfaces, and their respective affinities to ... More
Absolute requirement of cyclic nucleotide in the activation of the G141Q mutant cAMP receptor protein from Escherichia coli.
AuthorsCheng X, Lee JC
JournalJ Biol Chem
PubMed ID7983007
'cAMP receptor protein (CRP), when interacting with cAMP, controls the expression of a network of catabolite-sensitive genes in Escherichia coli. To understand the molecular events that lead to the activation of CRP, a combined approach of site-directed mutagenesis and thermodynamic analysis was employed to study a member of a specific ... More
Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies.
AuthorsZhang Z, Spiering MM, Trakselis MA, Ishmael FT, Xi J, Benkovic SJ, Hammes GG
JournalProc Natl Acad Sci U S A
PubMed ID15728347
'Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with ... More
Topography of the human factor VIII-von Willebrand factor complex.
AuthorsFay PJ, Smudzin TM
JournalJ Biol Chem
PubMed ID2108154
'Factor VIII circulates in noncovalent complex with von Willebrand factor (vWf). The topography of this complex was evaluated by fluorescence energy transfer using factor VIII subunits modified with N-(1-pyrenyl)maleimide (NPM; fluorescence donor) and vWf-derived fragments modified with 7-diethylamino-3-[4''-maleimidylphenyl]-4-methyl coumarin (CPM; fluorescence acceptor). Results from a previous study indicated an interfactor ... More
Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.
AuthorsOser A, Roth WK, Valet G
JournalNucleic Acids Res
PubMed ID3344204
'A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation ... More
Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.
AuthorsTao T, Gong BJ, Leavis PC
JournalScience
PubMed ID2138356
'The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between ... More
Identification of a major low-molecular-mass thiol of the trypanosomatid Crithidia fasciculata as ovothiol A. Facile isolation and structural analysis of the bimane derivative.
AuthorsSteenkamp DJ, Spies HS
JournalEur J Biochem
PubMed ID8033907
'An unidentified low-molecular-mass thiol, U23, previously detected as the 7-diethylamino-3-(4''-maleimidylphenyl)-4-methylcoumarin derivative in extracts of the trypanosome Crithidia fasciculata, was purified as the bimane derivative. Resonances attributable to U23 were discerned from those of the bimane label by comparison of the 1H- and 13C-NMR spectra of monobromobimane and U23-bimane. The complete ... More
Microscale fluorescent thermal stability assay for membrane proteins.
AuthorsAlexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC,
JournalStructure
PubMed ID18334210
'Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during ... More
Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I.
AuthorsLuo Y, Leszyk J, Qian Y, Gergely J, Tao T
JournalBiochemistry
PubMed ID10350487
'It has been proposed [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644], and confirmed by structural studies [Gagne et al. (1995) Nat. Struct. Biol. 2, 784-789], that the binding of Ca2+ to the triggering sites in troponin-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the ... More
Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence.
AuthorsGrossman SH
JournalBiochemistry
PubMed ID2765518
'Resonance energy transfer between the reactive thiols of rabbit muscle creatine kinase was evaluated. The reactive thiols are located at the active site, one occurring on each subunit of the dimeric protein that is known to be a constituent of the M-line structure of the myofibril. Transfer efficiency was evaluated ... More
Resonance energy transfer: methods and applications.
AuthorsWu P, Brand L
JournalAnal Biochem
PubMed ID8053542
'Resonance energy transfer is widely used in studies of biomolecular structure and dynamics. It provides information about distances on the order of 10 to 100 A and is thus suitable for investigating spatial relationships of interest in biochemistry. The information available from energy transfer studies has been enhanced by the ... More
Interdomain signaling in a two-domain fragment of the human glucocorticoid receptor.
AuthorsKumar R, Baskakov IV, Srinivasan G, Bolen DW, Lee JC, Thompson EB
JournalJ Biol Chem
PubMed ID10455143
'Studies of individual domains or subdomains of the proteins making up the nuclear receptor family have stressed their modular nature. Nevertheless, these receptors function as complete proteins. Studies of specific mutations suggest that in the holoreceptors, intramolecular domain-domain interactions are important for complete function, but there is little knowledge concerning ... More
The covalent maleimidobenzoyl-actin-myosin head complex. Cross-linking of the 50 kDa heavy chain region to actin subdomain-2.
AuthorsBertrand R, Derancourt J, Kassab R
JournalFEBS Lett
PubMed ID8200441
'We have identified the region of actin involved in the covalent coupling of maleimidobenzoyl-G-actin to the central 50 kDa segment of the myosin-S-1 heavy chain by analyzing the structure of the maleimidobenzoyl-G-actin-S-1 conjugate using selective proteolytic digestions, amino acid sequence determinations and novel cross-linking reactions between S-1 and different maleimidobenzoyl-G-actin ... More
Effect of ionic strength on the conformation of myosin subfragment 1-nucleotide complexes.
AuthorsPeyser YM, Ajtai K, Burghardt TP, Muhlrad A
JournalBiophys J
PubMed ID11463651
'The effect of ionic strength on the conformation and stability of S1 and S1-nucleotide-phosphate analog complexes in solution was studied. It was found that increasing concentration of KCl enhances the reactivity of Cys(707) (SH1 thiol) and Lys(84) (reactive lysyl residue) and the nucleotide-induced tryptophan fluorescence increment. In contrast, high KCl ... More
Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide.
AuthorsAllen RL, Olins DE
JournalChromosoma
PubMed ID6084583
'A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins ... More
ADP binding induces long-distance structural changes in the beta polypeptide of the chloroplast ATP synthase.
AuthorsMills DA, Seibold SA, Squier TC, Richter ML
JournalBiochemistry
PubMed ID7742314
'Binding of ADP to the beta polypeptide isolated from the catalytic F1 portion (CF1) of the chloroplast ATP synthase caused an increase of 10-20% in the steady state fluorescence intensity of fluorescent maleimides attached to the cysteine residue at position 63. Fluorescence lifetime distributions indicated that the beta polypeptide switched ... 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
An essential role for the extracellular domain of the Na,K-ATPase beta-subunit in cation occlusion.
AuthorsLutsenko S, Kaplan JH
JournalBiochemistry
PubMed ID8392370
'The role of the Na,K-ATPase beta-subunit in stabilization of ion-binding sites has been investigated. Treatment of the purified renal Na,K-ATPase with 0.25 M DTT at 40 degrees C for 1 h resulted in 50% loss of Rb occlusion, which correlates with partial reduction of S-S bridges in the extracellular portion ... More
The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol.
AuthorsSinha ND, Cook RM
JournalNucleic Acids Res
PubMed ID3362678
'Syntheses of H-phosphonate salts (4a-e) of N/S-protected alcohols such as 6-aminohexan-1-ol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol are described using 2-chloro-5,6-benzo-1,3,2-phosphorin-4-one (2) as the phosphonylating agent. The H-phosphonate salts (4a-e), in the presence of pivaloyl chloride or adamantoyl chloride as an activator, were coupled to the 5''-end of synthetic oligonucleotides on solid supports ... More
N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat.
AuthorsLutsenko S, Petrukhin K, Cooper MJ, Gilliam CT, Kaplan JH
JournalJ Biol Chem
PubMed ID9228074
'N-terminal domains of the Wilson''s and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding ... More
Application of a fluorescent histone acetyltransferase assay to probe the substrate specificity of the human p300/CBP-associated factor.
AuthorsTrievel RC, Li FY, Marmorstein R
JournalAnal Biochem
PubMed ID11112280
'Histone N-acetyltransferases (HATs) are a group of enzymes which acetylate specific lysine residues in the N-terminal tails of nucleosomal histones to promote transcriptional activation. Recent structural and enzymatic work on the GCN5/PCAF HAT family has elucidated the structure of their catalytic domain and mechanism of histone acetylation. However, the substrate ... More
Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca2+ release mechanism.
AuthorsZable AC, Favero TG, Abramson JJ
JournalJ Biol Chem
PubMed ID9054399
'In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Reduced glutathione (GSH) inhibited Ca2+-stimulated [3H]ryanodine binding to the sarcoplasmic reticulum and inhibited the single-channel gating activity of the reconstituted Ca2+ release channel. The effects of GSH on ... More
Regulation of the cGMP phosphodiesterase in bovine rod outer segments. Use of resonance energy transfer to distinguish between associative and dissociative activation mechanisms.
AuthorsErickson JW, Cerione RA
JournalJ Biol Chem
PubMed ID8381426
'Resonance energy transfer was used to monitor the interactions of the gamma subunit of the cyclic GMP phosphodiesterase (gamma PDE) with the alpha and beta subunits, which together comprise the catalytic core of the enzyme (alpha beta PDE). An iodoacetamidofluorescein (IAF)-labeled alpha beta PDE (IAF-PDEt) served as the acceptor species ... More
Mode of selectivity in cyclic AMP receptor protein-dependent promoters in Escherichia coli.
AuthorsPyles EA, Lee JC
JournalBiochemistry
PubMed ID8573570
'Escherichia coli cAMP receptor protein (CRP) controls more than 20 genes. There are significant differences in the promoter regions in these genes. Thus, an elucidation of the mechanism of CRP action requires knowledge about the mode of selectivity in these promoters. An earlier study [Heyduk, T., & Lee, J. C. ... More
GroEL stimulates protein folding through forced unfolding.
AuthorsLin Z, Madan D, Rye HS,
JournalNat Struct Mol Biol
PubMed ID18311152
'Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of ... 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
Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.
AuthorsWu J, Frillingos S, Voss J, Kaback HR
JournalProtein Sci
PubMed ID7756985
'By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4''-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances ... More
Interactions of cytochrome P450 2B4 with NADPH-cytochrome P450 reductase studied by fluorescent probe.
AuthorsDavydov DR, Knyushko TV, Kanaeva IP, Koen YM, Samenkova NF, Archakov AI, Hui Bon Hoa G
JournalBiochimie
PubMed ID9010602
'A new method for monitoring the formation of the cytochrome P450 complexes with NADPH-cytochrome P450 reductase (NCPR) is introduced. The method is based on the quenching of fluorescence of NCPR labelled with 7-ethylamino-3-(4''-maleimidilphenyl)-4-methylcoumarin maleimide (CPM). In a monomerized soluble reconstituted system in the absence of phospholipid, cytochrome P450 2B4 and ... More
Spatial organization of template polynucleotides on the ribosome determined by fluorescence methods.
AuthorsBakin AV, Borisova OF, Shatsky IN, Bogdanov AA
JournalJ Mol Biol
PubMed ID1717698
'The spatial organization of template polynucleotides on the ribosome and the dynamics of their interaction with 30 S subunits have been studied by fluorescence spectroscopy. The topography of the mRNA in the ribosome has been determined using singlet-singlet energy transfer. This method has allowed us to estimate distances between donors ... More
Flippase activity detected with unlabeled lipids by shape changes of giant unilamellar vesicles.
AuthorsPapadopulos A, Vehring S, López-Montero I, Kutschenko L, Stöckl M, Devaux PF, Kozlov M, Pomorski T, Herrmann A
JournalJ Biol Chem
PubMed ID17369612
'Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool ... More
A structural change in the kinesin motor protein that drives motility.
AuthorsRice S, Lin AW, Safer D, Hart CL, Naber N, Carragher BO, Cain SM, Pechatnikova E, Wilson-Kubalek EM, Whittaker M, Pate E, Cooke R, Taylor EW, Milligan RA, Vale RD
JournalNature
PubMed ID10617199
'Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region ... More
Electrostatic interaction of a K+ channel RCK domain with charged membrane surfaces.
AuthorsPtak CP, Cuello LG, Perozo E
JournalBiochemistry
PubMed ID15628846
'In a subset of K(+) channels, gating is regulated through the direct binding of ligands by large cytoplasmic RCK domains. To further investigate the role of the RCK domain, we have begun the biochemical characterization of a two-transmembrane segment, RCK domain-containing channel from Methanococcus jannaschii, MjK2, by testing its general ... More
Multiple end labeling of oligonucleotides with terbium chelate-substituted psoralen for time-resolved fluorescence detection.
AuthorsOser A, Collasius M, Valet G
JournalAnal Biochem
PubMed ID2085175
'A new procedure for the photochemical functionalization and the subsequent nonradioactive labeling of synthetic oligonucleotides with psoralen derivatives was developed where a double-stranded poly(A-T) tail is attached to the 5''- or 3''-end of the oligonucleotide to be labeled. The double-stranded poly(A-T) tail is covalently crosslinked by psoralen molecules which carry ... More
Fluorometric assay protocol for protease-catalyzed transesterification reactions in organic solvents.
AuthorsHan MS, Jung SO, Kim MJ, Kim DH
JournalJ Org Chem
PubMed ID15074938
'A flourometric assay protocol for a subtilisin-catalyzed transesterification reaction in n-hexane has been developed. The method makes use of a Michael acceptor that forms a fluorescent adduct with thiophenol, one of the products generated in the transesterification reaction. The method may be employed for screening a biocatalyst useful for transesterification ... More
Measurement of cyst(e)amine in physiological samples by high performance liquid chromatography.
AuthorsGarcia RA, Hirschberger LL, Stipanuk MH
JournalAnal Biochem
PubMed ID3394941
'Two methods for measurement of cyst(e)amine in physiological samples are described. One method involves reduction of disulfides present in the sample with tributylphosphine, reversed phase chromatography of thiols, and electrochemical detection of cysteamine and other thiols. The other method involves reduction of disulfides with dithiothreitol, derivatization of thiols with 7-diethylamino-3-(4''-maleimidylphenyl)-4-methylcoumarin, ... More
Structural mapping of Fc receptor bound immunoglobulin E: proximity to the membrane surface of the antibody combining site and another site in the Fab segments.
AuthorsBaird B, Holowka D
JournalBiochemistry
PubMed ID4084517
'Resonance energy-transfer methods have been used to investigate the structure of immunoglobulin E (IgE) bound to its high-affinity receptor on plasma membrane vesicles derived from rat basophilic leukemia cells. The structural mapping of receptor-bound IgE was initiated in an earlier study [Holowka, D., & Baird, B. (1983) Biochemistry 22, 3475], ... More
Formation and properties of smooth muscle myosin 20-kDa light chain-skeletal muscle myosin hybrids and photocrosslinking from the maleimidylbenzophenone-labeled light chain to the heavy chain.
AuthorsRajasekharan KN, Morita JI, Mayadevi M, Ikebe M, Burke M
JournalArch Biochem Biophys
PubMed ID1832844
'Experimental conditions which permit the exchange of smooth muscle 20-kDa light chain into skeletal muscle myosin are described. The hybridization does not result in the regulation of actin-activated ATPase activity of the hybrid myosin by smooth light chain phosphorylation. Further, the KCl dependence of the Mg-ATPase activity of the hybrid ... More
Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases.
AuthorsPicking WD, Kudlicki W, Kramer G, Hardesty B, Vandenheede JR, Merlevede W, Park IK, DePaoli-Roach A
JournalBiochemistry
PubMed ID1657143
'Phosphatase inhibitor 2 was mutagenized and expressed in Escherichia coli to produce a protein with a single cysteinyl residue at position 129. The newly introduced sulfhydryl group was labeled with a maleimide derivative of coumarin (CPM). The resulting fluorescent inhibitor 2 molecule (CPM-I2) retains biological activity and binds to the ... More
Chromogenic and fluorogenic assays for the lactonase activity of serum paraoxonases.
AuthorsKhersonsky O, Tawfik DS
JournalChembiochem
PubMed ID16329153
Interaction of creatine kinase from monkey brain with substrate: analysis of kinetics and fluorescence polarization.
AuthorsGrossman SH
JournalJ Neurochem
PubMed ID6875561
New fluorochromes for thiols: maleimide and iodoacetamide derivatives of a 3-phenylcoumarin fluorophore.
AuthorsSippel TO
JournalJ Histochem Cytochem
PubMed ID7019305
N-(4-(7 - Diethylamino - methylcoumarin - 3 yl)phenyl)maleimide (CPM) and the corresponding iodoacetamide are described. When applied to sections at pH 6 and 9, respectively, the two fluorogens are very similar in their reactivity and selectivity toward thiols with which they form adducts having much the same bright blue fluorescence. ... More
Skeletal muscle ryanodine receptor is a redox sensor with a well defined redox potential that is sensitive to channel modulators.
AuthorsXia R, Stangler T, Abramson JJ
JournalJ Biol Chem
PubMed ID10952995
Hyperreactive sulfhydryl groups associated with the Ca(2+) release protein from sarcoplasmic reticulum are shown to have a well defined reduction potential that is sensitive to the cellular environment. Ca(2+) channel activators lower the redox potential of the ryanodine receptor, which favors the oxidation of thiols and the opening of the ... More
Characterization of the resonance energy transfer couple coumarin-BODIPY and its possible applications in protein-lipid research.
AuthorsKeller RC, Silvius JR, De Kruijff B
JournalBiochem Biophys Res Commun
PubMed ID7864836
In our search for suitable resonance energy transfer (RET) couples for studying protein-lipid interactions, the promising couple coumarin-BODIPY was found and characterized. We characterized the RET from the donor coumarin to two different dipyrrometheneboron difluoride (BODIPY)-labeled phospholipid analogs both experimentally and theoretically. Calculations using the spectral overlap revealed a Förster ... More
Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit.
AuthorsColvert KK, Mills DA, Richter ML
JournalBiochemistry
PubMed ID1533153
The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of ... 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
A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme a production.
AuthorsChung CC, Ohwaki K, Schneeweis JE, Stec E, Varnerin JP, Goudreau PN, Chang A, Cassaday J, Yang L, Yamakawa T, Kornienko O, Hodder P, Inglese J, Ferrer M, Strulovici B, Kusunoki J, Tota MR, Takagi T,
JournalAssay Drug Dev Technol
PubMed ID18452391
Abstract: Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of ... More
Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer.
AuthorsNishino TG, Kitano K, Kojima K, Ogishima T, Ito A, Kitada S,
JournalJ Biochem
PubMed ID17426154
Mitochondrial processing peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a ... 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
Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli.
AuthorsCzworkowski J, Odom OW, Hardesty B
JournalBiochemistry
PubMed ID2029524
Short RNAs (25-36 nucleotides in length) with sequences of the translational initiation region of bacteriophage R17 protein A mRNA were produced by chemical and in vitro transcription techniques and labeled at their 5' or 3' ends with fluorescent probes. The interaction of these labeled RNAs with the 30S subunit of ... More
Fluorometric quantitation of cellular and nonprotein thiols.
AuthorsAyers FC, Warner GL, Smith KL, Lawrence DA
JournalAnal Biochem
PubMed ID3706721
A microfluorometric assay for thiols has been developed using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM). The technique may be used to quantitate either cellular or plasma thiols over a range of 0.01 to 3.0 nmol and may be used with as few as 1-3 X 10(5) cells giving highly proportional and ... More
In situ fluorescent visualization of nucleolar organizer region-associated proteins with a thiol reagent.
AuthorsMéhes G, Kálmán E, Pajor L
JournalJ Histochem Cytochem
PubMed ID8354881
Nucleolar organizer regions (NORs) are nucleolus-forming rDNA loops associated with argyrophil proteins, the amount of which varies according to the proliferative state of the cell. It has been presumed that the nucleolar protein-related thiol groups may have a role in selective silver staining. We investigated the nuclear thiol distribution with ... More
Dimeric association of Escherichia coli RNA polymerase alpha subunits, studied by cleavage of single-cysteine alpha subunits conjugated to iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate.
AuthorsMiyake R, Murakami K, Owens JT, Greiner DP, Ozoline ON, Ishihama A, Meares CF
JournalBiochemistry
PubMed ID9477962
Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with ... 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
Microfluorometric analysis of protein thiol groups with a coumarinylphenylmaleimide.
AuthorsSippel TO
JournalJ Histochem Cytochem
PubMed ID7320496
Characteristics of thiol adducts formed by the fluorogenic maleimide, N-(4-(7-diethylamino-4-methylcoumarin-3-yl) phenyl) maleimide (CPM), (Sippel: J Histochem Cytochem 29:314, 1981) were determined in solution, on the epithelia in beef cornea paraffin sections, and with an egg white model. Absorption was found maximal at 385-390 nm with a molar absorbancy of not ... 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
Lysosomal prenylcysteine lyase is a FAD-dependent thioether oxidase.
Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached via a thioether bond to a cysteine residue at or near their C terminus. As prenylated proteins comprise up to 2% of the total protein in eukaryotic cells, and the thioether bond is a stable modification, ... More
Molecular movements in the actomyosin complex: F-actin-promoted internal cross-linking of the 25- and 20-kDa heavy chain fragments of skeletal myosin subfragment.
AuthorsBertrand R, Derancourt J, Kassab R
JournalBiochemistry
PubMed ID1457419
We describe, for the first time, the F-actin-promoted changes in the spatial relationship of strands in the NH2-terminal 25-kDa and COOH-terminal 20-kDa heavy chain fragments of the skeletal myosin subfragment 1 (S-1), detected by their exclusive chemical cross-linking in the rigor F-actin-S-1 complex with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). Quantitative ... More
Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.
AuthorsHeyduk T, Lee JC
JournalProc Natl Acad Sci U S A
PubMed ID2155424
A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein ... More
Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP).
AuthorsLewis CT, Seyer JM, Carlson GM
JournalJ Biol Chem
PubMed ID2909519
Phosphoenolpyruvate carboxykinase from the cytosol of rat liver has 13 cysteines, at least one of which is known to be very reactive and essential for catalytic activity (Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 17, 5329-5338). In order to identify the essential cysteine, this enzyme was ... 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
Conformational changes in the Escherichia coli ATP synthase (ECF1F0) monitored by nucleotide-dependent differences in the reactivity of Cys-87 of the gamma subunit in the mutant betaGlu-381 --> Ala.
AuthorsFeng Z, Aggeler R, Haughton MA, Capaldi RA
JournalJ Biol Chem
PubMed ID8663500
Cys-87, one of two intrinsic cysteines of the gamma subunit of the Escherichia coli ATP synthase (ECF1F0), is in a short segment of this subunit that binds to the bottom domain of a beta subunit close to a glutamate (Glu-381). Cys-87 was unreactive to maleimides under all conditions in wild-type ... More
SecA, the peripheral subunit of the Escherichia coli precursor protein translocase, is functional as a dimer.
AuthorsDriessen AJ
JournalBiochemistry
PubMed ID8241173
SecA, the peripheral ATPase domain of the Escherichia coli precursor protein translocase, was denatured in 6 M guanidine hydrochloride. Circular dichroism and intrinsic tryptophan fluorescence spectra revealed that the protein is transformed into a random-coil configuration. Upon dilution of the chaotropic agent, SecA refolds into its native, functional conformation as ... More
Simple methods for the detection and quantification of thiols from Crithidia fasciculata and for the isolation of trypanothione.
AuthorsSteenkamp DJ
JournalBiochem J
PubMed ID8503857
Methods for the qualitative and quantitative analysis of thiols by means of the fluorogenic reagent 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin are described, with particular reference to the trypanosomatid metabolites glutathionylspermidine (GSH-spermidine) and trypanothione. Second-order rate constants for the derivatization of seven different thiols under defined experimental conditions and at 21 degrees C varied between ... More
The importance of the N-terminal segment for DnaJ-mediated folding of rhodanese while bound to ribosomes as peptidyl-tRNA.
Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein ... More
The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.
AuthorsWang J, de Montellano PR
JournalJ Biol Chem
PubMed ID12626517
Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. ... More
Functional and structural heterogeneity of the DNA binding site of the Escherichia coli primary replicative helicase DnaB protein.
AuthorsJezewska MJ, Rajendran S, Bujalowski W
JournalJ Biol Chem
PubMed ID9535894
The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is ... More
Structural mapping of the epsilon-subunit of mitochondrial H(+)-ATPase complex (F1).
AuthorsGabellieri E, Strambini GB, Baracca A, Solaini G
JournalBiophys J
PubMed ID9083686
Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the ... More
Inositol 1,4,5-trisphosphate receptor/Ca2+ channel modulatory role of chromogranin A, a Ca2+ storage protein of secretory granules.
AuthorsYoo SH, Jeon CJ
JournalJ Biol Chem
PubMed ID10748130
The secretory granules of neuroendocrine cells, which contain large amounts of Ca(2+) and chromogranins, have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)), indicating the IP(3)-sensitive intracellular Ca(2+) store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory ... More
Structural map of the dicyclohexylcarbodiimide site of chloroplast coupling factor determined by resonance energy transfer.
AuthorsMitra B, Hammes GG
JournalBiochemistry
PubMed ID2525921
Fluorescence resonance energy-transfer measurements were made on the membrane-bound chloroplast coupling factor. The distances from the N,N'-dicyclohexylcarbodiimide-binding site on the membrane-bound portion of the enzyme (CF0) to the vesicle surface and to two sulfhydryl sites on the gamma-polypeptide were determined. The dicyclohexylcarbodiimide-binding site was labeled with the fluorescent species N-cyclohexyl-N'-pyrenylcarbodiimide. ... More
Physical studies on interaction of transcription activator and RNA-polymerase: fluorescent derivatives of CRP and RNA polymerase.
AuthorsHeyduk E, Heyduk T
JournalCell Mol Biol Res
PubMed ID8312976
Protein-protein interactions between cAMP receptor protein (CRP) and RNA polymerase (RNAP) have been proposed to be essential in RNAP activation by CRP in type I promoters. These two proteins were shown to interact in solution in the absence of promoter DNA (Heyduk et al., 1993). In this report we describe ... More
Inhibitors of kinesin activity from structure-based computer screening.
AuthorsHopkins SC, Vale RD, Kuntz ID
JournalBiochemistry
PubMed ID10704233
Kinesin motor proteins use ATP hydrolysis for transport along microtubules in the cell. We sought to identify small organic ligands to inhibit kinesin's activity. Candidate molecules were identified by computational docking of commercially available compounds using the computer program DOCK. Compounds were docked at either of two sites, and a ... More
Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.
AuthorsFavero TG, Webb J, Papiez M, Fisher E, Trippichio RJ, Broide M, Abramson JJ
JournalJ Appl Physiol
PubMed ID12626470
We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the ... More
Solution studies on the structure of bent DNA in the cAMP receptor protein-lac DNA complex.
AuthorsHeyduk T, Lee JC
JournalBiochemistry
PubMed ID1606140
Cyclic AMP receptor protein is involved in the regulation of more than 20 genes. A step in the mechanism of activation of transcription is to induce a significant bending of the DNA upon complex formation between specific DNA and the protein. The induced DNA bending and a structure of the ... More