Fluorescence energy transfers between points in acto-subfragment-1 rigor complex.
AuthorsMiki M, Wahl P
JournalBiochim Biophys Acta
PubMed ID6487641
Fluorescence energy transfer was measured by time-resolved and steady-state fluorimetry in order to investigate the spatial relationships between the nucleotide binding site of actin, the Cys-373 residue of actin, and the SH1 of myosin subfragment-1 in the rigor complex of acto-subfragment-1. N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to the Cys-373 of actin or ... More
Bioluminescence resonance energy transfer from aequorin to a fluorophore: an artificial jellyfish for applications in multianalyte detection.
AuthorsDeo SK, Mirasoli M, Daunert S
JournalAnal Bioanal Chem
PubMed ID15731912
'In nature, the green light emission observed in the jellyfish Aequorea victoria is a result of a non-radiative energy transfer from the excited-state aequorin to the green fluorescent protein. In this work, we have modified the photoprotein aequorin by attaching selected fluorophores at a unique site on the protein. This ... More
Characterization of troponin-C interactions in skinned barnacle muscle: comparison with troponin-C from rabbit striated muscle.
AuthorsGordon AM, Qian Y, Luo Z, Wang CK, Mondares RL, Martyn DA
JournalJ Muscle Res Cell Motil
PubMed ID9429158
'Previously it was shown that when troponin-C (TnC) is extracted from barnacle myofibrillar bundles they lose their Ca2+ sensitivity, which can be restored by adding back barnacle TnC (either isoform, BTnC1 or BTnC2). Thus barnacle muscle shows thin filament regulation, as does rabbit psoas skeletal muscle. In this paper we ... 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
Complexes of myosin subfragment-1 with adenosine diphosphate and phosphate analogs: probes of active site and protein conformation.
AuthorsPhan BC, Cheung P, Stafford WF, Reisler E
JournalBiophys Chem
PubMed ID8672721
'Previous work has revealed phosphate-dependent differences in the complexes formed from myosin subfragment-1 with adenosine diphosphate (S1.ADP) and aluminum fluoride (AlF4-) or beryllium fluoride (BeFx) [Phan and Reisler, Biophys. J., 66 (1994) A78], with the former resembling more the S1**.ADP.Pi state while the latter resembles more the S1.ATP state. In ... More
Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C.
AuthorsDupont DM, Blouse GE, Hansen M, Mathiasen L, Kjelgaard S, Jensen JK, Christensen A, Gils A, Declerck PJ, Andreasen PA, Wind T
JournalJ Biol Chem
PubMed ID17018527
'Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is ... More
Kinetics of binding of caldesmon to actin.
AuthorsChalovich JM, Chen YD, Dudek R, Luo H
JournalJ Biol Chem
PubMed ID7730374
'The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using ... More
Identification of the functionally relevant calmodulin binding site in smooth muscle caldesmon.
AuthorsZhuang S, Wang E, Wang CL
JournalJ Biol Chem
PubMed ID7650012
'The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD''s inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and ... More
Calmodulin levels are dynamically regulated in living vascular smooth muscle cells.
AuthorsHulvershorn J, Gallant C, Wang CA, Dessy C, Morgan KG
JournalAm J Physiol Heart Circ Physiol
PubMed ID11179093
'The total unbound calmodulin (i.e., not bound to target proteins) level in living smooth muscle cells from the ferret portal vein was monitored with a low-affinity, calmodulin-binding peptide tagged with an environmentally sensitive fluorophore. GS17C, a previously characterized peptide, from the calmodulin-binding domain of caldesmon was tagged with iodoacetyl nitrobenz-2-oxa-1,3-diazole ... More
'Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share ... More
A calmodulin-binding peptide of caldesmon.
AuthorsZhan QQ, Wong SS, Wang CL
JournalJ Biol Chem
PubMed ID1939204
'Caldesmon is a major actin-binding protein identified in smooth muscle and many non-muscle cells. It also interacts with calmodulin and a number of other acidic proteins. We have shown previously that the polypeptide stretch from Val629 to Ser666 near the C terminus contains a calmodulin binding site (Wang, C.-L. A., ... More
Effects of SH1 and SH2 modifications on myosin: similarities and differences.
AuthorsBobkova EA, Bobkov AA, Levitsky DI, Reisler E
JournalBiophys J
PubMed ID9916031
'The properties of myosin modified at the SH2 group (Cys-697) were studied and compared with the previously reported properties of myosin modified at the SH1 group (Cys-707). 4-[N-[(iodoacetoxy)ethyl]-N methylamino]-7-nitrobenz-2-oxa-1, 3-diazole (IANBD) was used for selective modification of the SH2 group on myosin. SH2-labeled heavy meromyosin (SH2-HMM), similar to SH1-labeled HMM ... More
Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione.
AuthorsLiang JN, Pelletier MR
JournalExp Eye Res
PubMed ID3653289
'Mixed disulfide was formed through thiol-disulfide exchange reaction of lens crystallin with oxidized glutathione (GSSG). The reaction was monitored by isoelectric focusing (IEF) and DTNB [5,5''-dithiobis(2-nitrobenzoic acid)] assay. The effects on protein conformation were studied by circular dichroism (CD) and fluorescence. The DTNB shows 22% and 49% decrease of SH ... More
Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin.
AuthorsKorman VL, Tobacman LS
JournalJ Biol Chem
PubMed ID10428784
'Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen ... More
Distance between nucleotide site and cysteine-373 of G-actin by resonance energy transfer measurements.
AuthorsCheung HC, Liu BM
JournalJ Muscle Res Cell Motil
PubMed ID6715528
'The distance between the nucleotide site and the reactive cysteine-373 of G-actin was determined from resonance energy transfer measurements by using 1,N6-ethenoadenosine triphosphate (epsilon ATP) as the donor and 4-[N-(iodoacetoxy)ethyl N methyl]amino 7 nitrobenz 2 oxa 1,3 diazole covalently attached to the sulphydryl group as acceptor. The quenching of the ... More
Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.
AuthorsGether U, Lin S, Ghanouni P, Ballesteros JA, Weinstein H, Kobilka BK
JournalEMBO J
PubMed ID9362488
'Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that ... 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
Fluorescence studies on tryptophan and sulfhydryl group changes of bovine lens crystallins in a photodynamic system.
AuthorsAndley UP, Chapman SF, Chylack LT
JournalCurr Eye Res
PubMed ID4042665
'Conformational changes in the three crystallins alpha-, beta-, and gamma- in a singlet-oxygen generating system were investigated by fluorescence studies of tryptophan and covalently-bound sulfhydryl probe 4-[(N-iodoacetoxy)N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD). Upon excitation at 295 nm, the tryptophan emission maxima of the crystallins were red-shifted by irradiation with visible light in the presence ... More
Synthesis, location, and lateral mobility of fluorescently labeled ubiquinone 10 in mitochondrial and artificial membranes.
AuthorsRajarathnam K, Hochman J, Schindler M, Ferguson-Miller S
JournalBiochemistry
PubMed ID2742832
'To explore the influence of the long isoprene chain of ubiquinone 10 (UQ) on the mobility of the molecule in a phospholipid bilayer, we have synthesized a fluorescent derivative of the head-group moiety of UQ and measured its lateral diffusion in inner membranes of giant mitochondria and in large unilamellar ... More
Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states.
AuthorsTrayer HR, Trayer IP
JournalBiochemistry
PubMed ID2972314
'Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography ... More
Activation and desensitization of Torpedo acetylcholine receptor: evidence for separate binding sites.
AuthorsDunn SM, Raftery MA
JournalProc Natl Acad Sci U S A
PubMed ID6960348
'The acetylcholine receptor from Torpedo californica was labeled by reaction with the fluorescent probe 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole without apparent effect on its in vitro ligand binding and functional properties. Addition of acetylcholine or carbamoylcholine to the labeled-receptor preparations enhanced the fluorescence of the bound probe, and this effect was specific for agonists ... More
The rate of MgADP binding to and dissociation from acto-S1.
AuthorsBorejdo J, Ando T, Burghardt TP
JournalBiochim Biophys Acta
PubMed ID3872135
'The rate of binding and dissociation of MgADP from its ternary complex with actin and S1 was measured by following the extent to which fixed concentrations of MgADP slow down MgATP-induced dissociation of acto-S1. The solution of the equations describing this process shows that at any MgADP concentration the apparent ... More
Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors.
AuthorsSimard JR, Getlik M, Grütter C, Pawar V, Wulfert S, Rabiller M, Rauh D,
JournalJ Am Chem Soc
PubMed ID19572644
'Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less ... More
NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants.
AuthorsSengupta S, Banerjee S, Chakrabarti G, Mahapatra PK, Roy S, Bhattacharyya B
JournalBiochemistry
PubMed ID10694388
'Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can ... More
Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli DNA polymerase I.
AuthorsAllen DJ, Benkovic SJ
JournalBiochemistry
PubMed ID2692712
'Resonance energy transfer was used to determine separation distances between fluorescent derivatives of substrates for Klenow fragment and a unique sulfhydryl, cysteine 907, on the enzyme. Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates (dNTP), and deoxynucleotide monophosphates (dNMP), modified with aminonaphthalenesulfonates (ANS), served as energy-transfer donors to the fluorophore used ... More
Tissue-type plasminogen activator and urokinase: differences in the reaction pattern with the active-site titrant 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride.
AuthorsGeiger M, Binder BR
JournalBiochim Biophys Acta
PubMed ID3103686
'The applicability of 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride (MUGB) as active-site titrant for tissue-type plasminogen activator (t-PA) was studied in comparison to urokinase. Although t-PA was capable of cleaving MUGB, active-site titration of t-PA (one-chain form as well as two-chain form) with MUGB was not possible, whereas MUGB titration of urokinase could be ... More
Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes.
AuthorsStan-Lotter H, Bragg PD
JournalEur J Biochem
PubMed ID2876891
'In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4''-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility ... More
Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers.
AuthorsAjtai K, Burghardt TP
JournalBiochemistry
PubMed ID2524213
'We describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge ... More
Characterization of calponin binding to actin.
AuthorsLu FW, Freedman MV, Chalovich JM
JournalBiochemistry
PubMed ID7547921
'Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding ... More
The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors.
'Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions ... More
Caldesmon reduces the apparent rate of binding of myosin S1 to actin-tropomyosin.
AuthorsSen A, Chen YD, Yan B, Chalovich JM
JournalBiochemistry
PubMed ID11341841
'Equilibrium measurements of the rate of binding of caldesmon and myosin S1 to actin-tropomyosin from different laboratories have yielded different results and have led to different models of caldesmon function. An alternate approach to answering these questions is to study the kinetics of binding of both caldesmon and S1 to ... More
Separate sites of low and high affinity for agonists on Torpedo californica acetylcholine receptor.
AuthorsDunn SM, Conti-Tronconi BM, Raftery MA
JournalBiochemistry
PubMed ID6860645
'We have studied alkylation of the membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electric organ by the cholinergic agonist bromo-acetylcholine (BrAcCh). Following reduction of the AcChR with dithiothreitol (DTT) under strictly controlled conditions, a single class of binding sites was covalently labeled by BrAcCh. The extent of alkylation was dependent ... More
Caldesmon has two calmodulin-binding domains.
AuthorsWang CL, Wang LW, Lu RC
JournalBiochem Biophys Res Commun
PubMed ID2757638
'Chicken gizzard caldesmon was cleaved with chymotrypsin or CNBr, and the calmodulin-binding fragments were isolated using an affinity column. Limited chymotryptic digestion gives rise to a 38 kDa calmodulin-binding fragment (CT40) as described previously (Szpacenko, A. & Dabrowska, R., FEBS Lett. 202, 182-186, 1986; Fujii, T., Imai, M., Rosenfeld, G. ... More
Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A.
AuthorsLawrence DA, Olson ST, Muhammad S, Day DE, Kvassman JO, Ginsburg D, Shore JD
JournalJ Biol Chem
PubMed ID10681574
'The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the ... More
Assessing adrenergic receptor conformation using chemically reactive fluorescent probes.
AuthorsJensen AD, Gether U
JournalMethods Mol Biol
PubMed ID10685422
Examination of ligand-induced conformational changes in the beta 2-adrenergic receptor by fluorescence spectroscopy.
AuthorsKobilka BK, Gether U
JournalAdv Pharmacol
PubMed ID9327941
Independent sites of low and high affinity for agonists on Torpedo californica acetylcholine receptor.
AuthorsConti-Tronconi BM, Dunn SM, Raftery MA
JournalBiochem Biophys Res Commun
PubMed ID7126198
Resonance energy transfer between catalytic sites of bovine liver uridine diphosphoglucose dehydrogenase.
AuthorsFranzen JS, Marchetti PS, Feingold DS
JournalBiochemistry
PubMed ID7470452
Structure of actin observed by fluorescence resonance energy transfer spectroscopy.
AuthorsMiki M, O'Donoghue SI, Dos Remedios CG
JournalJ Muscle Res Cell Motil
PubMed ID1534564
Myosin structure. Proximity measurements by fluorescence energy transfer.
AuthorsHaugland RP
JournalJ Supramol Struct
PubMed ID127888
Conformational flexibility and structure of creatine kinase.
AuthorsHaugland RP
JournalJ Supramol Struct
PubMed ID1195743
The structural flexibility of creatine kinase has been investigated with the covalent hydrophobic probe 2-[4'-(2"-iodoacetamido) phenyl] aminonaphthalene-6-sulfonic acid (IAANS) which reacts at vastly different rates with the two subunits to give a protein conjugate with fluorescence characteristic of reaction with a site in a hydrophobic cleft. Binding of purine nucleotides ... More
Serpin-protease complexes are trapped as stable acyl-enzyme intermediates.
AuthorsLawrence DA, Ginsburg D, Day DE, Berkenpas MB, Verhamme IM, Kvassman JO, Shore JD
JournalJ Biol Chem
PubMed ID7592687
The serine protease inhibitors of the serpin family are an unusual group of proteins thought to have metastable native structures. Functionally, they are unique among polypeptide protease inhibitors, although their precise mechanism of action remains controversial. Conflicting results from previous studies have suggested that the stable serpin-protease complex is trapped ... 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
Engineering the maltose binding protein for reagentless fluorescence sensing.
AuthorsGilardi G, Zhou LQ, Hibbert L, Cass AE
JournalAnal Chem
PubMed ID7802263
This paper describes a mutant of the maltose binding protein (MBP) in which the serine residue at position 337 is replaced by a cysteine residue using site-directed mutagenesis. The mutant MBP has an approximately 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and ... More
Ca(2+)-dependence of structural changes in troponin-C in demembranated fibers of rabbit psoas muscle.
AuthorsAllen TS, Yates LD, Gordon AM
JournalBiophys J
PubMed ID1547328
The Ca(2+)-dependence of structural changes in troponin-C (TnC) has been detected by monitoring the fluorescence from TnC labeled at Methionine-25, in the NH2-terminal domain, with danzylaziridine (TnC-DANZ) and then exchanged for endogenous TnC in glycerinated single fibers. The fluorescence-pCa relation obtained from fibers stretched to a sarcomere length greater than ... More
Does actin bind to the ends of thin filaments in skeletal muscle?
AuthorsIshiwata S, Funatsu T
JournalJ Cell Biol
PubMed ID3880755
We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ... More
Examination of ligand-induced conformational changes in the beta2 adrenergic receptor.
AuthorsKobilka B, Gether U, Seifert R, Lin S, Ghanouni P
JournalLife Sci
PubMed ID9585127
The environmentally sensitive and cysteine reactive fluorescent probe, IANBD, was used to monitor ligand-induced structural changes in the beta2 adrenergic receptor (beta2AR) by fluorescent spectroscopy. We found that agonists caused a dose-dependent and reversible decrease in fluorescence from the purified IANBD-labeled beta2AR. This suggested that agonists promote a conformational change ... More
Fluorescent glucagon derivatives. II. The use of fluorescent glucagon derivatives for the study of receptor disposition in membranes.
AuthorsWard LD, Cantrill RC, Heithier H, Peters R, Helmreich EJ
JournalBiochim Biophys Acta
PubMed ID2844292
When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, ... More
Fluorescence study of the effects of aging and diabetes mellitus on human lens alpha-crystallin.
AuthorsLiang JN
JournalCurr Eye Res
PubMed ID3568749
Human alpha-crystallins were separated from fetal, young, senile nondiabetic and diabetic lenses. The effects of aging and diabetes mellitus were studied by fluorescence measurements, including emission maximum, quantum yield and polarization, using both intrinsic probes (tryptophan and non-tryptophan) and extrinsic probes [4-(N-iodoacetoxy)N-methylamino-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS)]. Results indicate that diabetic ... More
Resonance energy transfer determination of the distance between the four cysteine-364 residues in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
AuthorsAlvear M, Encinas MV, Herrera L, Cardemil E
JournalArch Biochem Biophys
PubMed ID8135532
Each of the four subunits of the Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase has one cysteine residue (Cys-364) that is protected against alkylation by MnATP and that is thought to be located at (or close to) the active site (M. Alvear, M. V. Encinas, S. Latshaw, R. G. Kemp, and E. Cardemil, ... More
Fluorescence titration and fluorescence stopped-flow studies of skeletal muscle troponin-nonpolymerizable tropomyosin complex.
AuthorsIio T, Nishio T, Kondo H
JournalJ Biochem (Tokyo)
PubMed ID3182770
The midpoint pCa value of the fluorescence titration curve of the complex of 2-[4'-iodoacetamido)anilino)-naphthalene-6-sulfonic acid-labeled troponin (IAANS-Tn) and nonpolymerizable tropomyosin (NPTm) was much larger than that for the complex of Tn containing dansylaziridine-labeled troponin C (DANZ-TnC) and NPTm. The midpoint was pCa 8.25 for the former protein and 6.80 for ... More
Adenosine 5'-(gamma-thiotriphosphate): an ATP analog that should be used with caution in muscle contraction studies.
AuthorsResetar AM, Chalovich JM
JournalBiochemistry
PubMed ID8519760
The slowly hydrolyzed ATP analog adenosine 5'-(gamma-thiotriphosphate) (ATP gamma S) has been used in many studies of the muscle motor protein myosin in order to form a stable "weak binding" state analogous to the actin-S1-ATP complex However, the results from studies using ATP gamma S do not always agree with ... More
Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors.
AuthorsRenard M, Belkadi L, Bedouelle H
JournalJ Mol Biol
PubMed ID12547199
The possibility of obtaining, from any antibody, a fluorescent conjugate which responds to the binding of the antigen by a variation of fluorescence, would be of great interest in the micro- and nano-analytical sciences. This possibility was explored with antibody mAb4E11, which is directed against the dengue virus and for ... More
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: revised amino acid sequence, site-directed mutagenesis, and microenvironment characteristics of cysteines 365 and 458.
AuthorsKrautwurst H, Encinas MV, Marcus F, Latshaw SP, Kemp RG, Frey PA, Cardemil E
JournalBiochemistry
PubMed ID7756267
Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine ... More
Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits.
AuthorsStan-Lotter H, Bragg PD
JournalEur J Biochem
PubMed ID2867900
The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble ... More
Troponin-tropomyosin: an allosteric switch or a steric blocker?
AuthorsResetar AM, Stephens JM, Chalovich JM
JournalBiophys J
PubMed ID12124285
The interaction of myosin subfragment 1 (S1) with actin-tropomyosin-troponin (regulated actin) is highly nucleotide dependent. The binding of S1 or S1-ADP (but not S1-ATP nor N,N'-rho-phenylenedimaleimide-modified S1-ATP) to regulated actin activates ATP hydrolysis even in the absence of Ca(2+). Investigations with S1 and S1-ADP have led to the idea that ... More
High-affinity actin-binding nebulin fragments influence the actoS1 complex.
AuthorsRoot DD, Wang K
JournalBiochemistry
PubMed ID11170442
Human nebulin fragments, NA3 and NA4, corresponding to individual superrepeats display high-affinity interactions with individual actin protomers in cosedimentation and solid-phase binding assays. Stoichiometric analysis of nebulin fragment-induced actin polymerization and inhibition of actin-activated S1 ATPase indicate that one superrepeat influences multiple actin molecules along the F-actin filament, consistent with ... More
Selective modification of the catalytic subunit of cAMP-dependent protein kinase with sulfhydryl-specific fluorescent probes.
AuthorsFirst EA, Taylor SS
JournalBiochemistry
PubMed ID2742857
The catalytic subunit of cAMP-dependent protein kinase contains only two cysteine residues, and the side chains of both Cys 199 and Cys 343 are accessible. Modification of the catalytic subunit by a variety of sulfhydryl-specific reagents leads to the loss of enzymatic activity. The differential reactivity of the two sulfhydryl ... More
Class-selective drug detection: fluorescently-labeled calmodulin as the biorecognition element for phenothiazines and tricyclic antidepressants.
AuthorsDouglass PM, Salins LL, Dikici E, Daunert S
JournalBioconjug Chem
PubMed ID12440852
A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants (TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as the recognition element. A calmodulin mutant containing a unique cysteine residue at position 109 on the protein was expressed in Escherichia coli. Following purification, the environment-sensitive, thiol-specific ... More
Interactions of peptides with DnaK and C-terminal DnaK fragments studied using fluorescent and radioactive peptides.
AuthorsZhang J, Walker GC
JournalArch Biochem Biophys
PubMed ID9705208
Monocysteine derivatives of Peptide C (KLIGVLSSLFRPK) were modified with N-((2-(acetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole (ANBD) to introduce a fluorescent probe. Five Peptide C derivatives-PepC-V5C-ANBD, PepC-L6C-ANBD, PepC-S7C-ANBD, PepC-S8C-ANBD, and PepC-L9C-ANBD-were then used to investigate the peptide-binding properties of DnaK. Introduction of the ANBD moiety at positions 8 and 9 of Peptide C yields peptides ... More
Dissecting the order of bacteriophage T4 DNA polymerase holoenzyme assembly.
Most biological organisms rely upon a DNA polymerase holoenzyme for processive DNA replication. The bacteriophage T4 DNA polymerase holoenzyme is composed of the polymerase enzyme and a clamp protein (the 45 protein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme. The 45 protein ... More
Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for MgATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase.
AuthorsFirst EA, Johnson DA, Taylor SS
JournalBiochemistry
PubMed ID2787168
The catalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., & Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic ... More
Multiple binding sites for agonists on Torpedo californica acetylcholine receptor.
AuthorsDunn SM, Raftery MA
JournalBiochemistry
PubMed ID7150556
The equilibrium and kinetic properties of agonist binding to the membrane-bound acetylcholine receptor from Torpedo californica have been measured by the fluorescence changes of a probe, 4-[[(iodoacetoxy)ethyl]methylamino]-7-nitro-2,1,3-benzoxadiazole, which was covalently bound to the receptor protein. Dissociation constants for the binding of several agonists have been measured in fluorescence titration experiments, ... More
Detection of a conserved alpha-helix in the kinase-docking region of the aspartate receptor by cysteine and disulfide scanning.
AuthorsBass RB, Falke JJ
JournalJ Biol Chem
PubMed ID9737956
The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range. Biochemical and genetic studies have implicated a ... More
A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation.
AuthorsPost PL, Trybus KM, Taylor DL
JournalJ Biol Chem
PubMed ID8175704
Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part ... More
Cooperative binding of myosin subfragment one to regulated actin as measured by fluorescence changes of troponin I modified with different fluorophores.
AuthorsGreene LE
JournalJ Biol Chem
PubMed ID3753701
Binding studies of myosin subfragment one (S-1) to regulated actin in the presence and absence of Ca2+ indicate that, as S-1 binds to regulated actin, tropomyosin-actin units undergo a cooperative transition from a weak to a strong S-1-binding form. Trybus and Taylor (Trybus, K.M., and Taylor, E.W. (1980) Proc. Natl. ... More
Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies.
AuthorsRenard M, Belkadi L, Hugo N, England P, Altschuh D, Bedouelle H
JournalJ Mol Biol
PubMed ID12051849
The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen ... More
Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations.
AuthorsLindahl P, Raub-Segall E, Olson ST, Björk I
JournalBiochem J
PubMed ID2049069
Papain was labelled by attachment of the fluorescent groups 2-(4'-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was ... More
Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition.
AuthorsMartyn DA, Freitag CJ, Chase PB, Gordon AM
JournalBiophys J
PubMed ID10049329
Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects ... More
Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers.
AuthorsFrey S, Tamm LK
JournalBiochem J
PubMed ID2176475
The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter ... More
The interaction of monomeric actin with two binding sites on Acanthamoeba actobindin.
AuthorsBubb MR, Lewis MS, Korn ED
JournalJ Biol Chem
PubMed ID1995634
Actobindin was previously shown to be an 88-residue polypeptide (Mr 9761) with an internal tandem repeat of 33-34 amino acids. Sedimentation equilibrium experiments have confirmed this Mr for native actobindin. Pyreneglyoxal-labeled actobindin had a similar Mr by sedimentation equilibrium analysis and bound to actin in a manner qualitatively similar to ... More
Relationship between regulated actomyosin ATPase activity and cooperative binding of myosin to regulated actin.
AuthorsGreene LE, Eisenberg E
JournalCell Biophys
PubMed ID2453286
The protein complex, troponin-tropomyosin, which is bound to the thin actin filament, regulates muscle contraction and relaxation. In the absence of Ca2+ the troponin-tropomyosin complex causes muscle to relax, whereas in the presence of Ca2+, contraction occurs. Biochemical studies have shown that the troponin-tropomyosin complex has a dual effect on ... More
Cholinergic binding sites on the pentameric acetylcholine receptor of Torpedo californica.
AuthorsDunn SM, Raftery MA
JournalBiochemistry
PubMed ID8102880
The binding of agonists, antagonists, and the acetylcholinesterase inhibitor, eserine, to the nicotinic acetylcholine receptor from Torpedo californica has been monitored by the fluorescence changes of two extrinsic probes that have been covalently attached to the receptor protein. Although both probes, 5-(iodoacetamido)salicylic acid (IAS) and 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diaz ol e (IANBD) react ... More
Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.
AuthorsHeithier H, Ward LD, Cantrill RC, Klein HW, Im MJ, Pollak G, Freeman B, Schiltz E, Peters R, Helmreich EJ
JournalBiochim Biophys Acta
PubMed ID2844291
The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), ... More
Models of the actin monomer and filament from fluorescence resonance-energy transfer.
AuthorsO'Donoghue SI, Hambly BD, dos Remedios CG
JournalEur J Biochem
PubMed ID1572360
We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficiency measurements into structural models which predict the relative positions of the chemical groups used in FRET. We used these algorithms to construct models of the actin monomer and filament derived solely from FRET measurements based on seven distinct loci. ... More
pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase.
AuthorsZhou X, Toney MD
JournalBiochemistry
PubMed ID9890912
The pH dependence of the steady-state kinetic parameters for the dialkylglycine decarboxylase-catalyzed decarboxylation-dependent transamination between 2-aminoisobutyrate (AIB) and pyruvate is presented. The pH dependence of methylation and DTNB modification reactions, and spectroscopic properties, is used to augment the assignment of the kinetic pKa's to specific ionizations. The coincidence of pKa ... More
Design and construction of glutamine binding proteins with a self-adhering capability to unmodified hydrophobic surfaces as reagentless fluorescence sensing devices.
AuthorsWada A, Mie M, Aizawa M, Lahoud P, Cass AE, Kobatake E
JournalJ Am Chem Soc
PubMed ID14692764
The chemically and genetically remodeling of proteins with ligand binding specificities can be utilized to synthesize various protein-based microsensors for detecting single biomolecules. Here, we describe the construction and characterization of fluorophore-labeled glutamine binding proteins (QBP) and derivatives coupled to the independently designed hydrophobic polypeptide (E12) that can adhere onto ... More
Kinetic studies of calcium binding to regulatory complexes from skeletal muscle.
AuthorsRosenfeld SS, Taylor EW
JournalJ Biol Chem
PubMed ID3965450
The kinetic mechanism of the binding and release of calcium by troponin and by the complexes troponin: tropomyosin, troponin:tropomyosin:actin, and troponin (TN)-tropomyosin (TM)-actin:myosin subfraction 1 (SF-1) was investigated using troponin labeled on the TN-I subunit with the fluorophore 4-(N-iodoac etoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole. The apparent association constant is five to 10 times smaller ... More
Kinetic studies of the cooperative binding of subfragment 1 to regulated actin.
AuthorsTrybus KM, Taylor EW
JournalProc Natl Acad Sci U S A
PubMed ID6938966
The transient-state kinetics of binding of myosin subfragment 1 (SF-1) to regulated actin in the presence and absence of Ca2+ were investigated. The binding of SF-1 to pure actin, to actin-tropomyosin (actin-TM), or to actin-tropomyosin-troponin (actin-TM-TN) in the presence of Ca2+ was kinetically the same. In each case, the light-scattering ... More
Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction
AuthorsBrenner B, Chalovich JM
JournalBiophys J
PubMed ID10545369
Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be ... More
Kinetic studies of calcium and magnesium binding to troponin C.
AuthorsRosenfeld SS, Taylor EW
JournalJ Biol Chem
PubMed ID3965449
The kinetic mechanism of calcium binding was investigated for the high-affinity calcium-magnesium sites of troponin C (TN-C), for the C-terminal fragment containing only the high-affinity sites (TR2) and for the TN-C:TN-I (where TN-I represents the inhibitory subunit of troponin) complex. Rate constants were measured by the change in fluorescence of ... More
Thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle
AuthorsBrenner B, Kraft T, Yu LC, Chalovich JM
JournalBiophys J
PubMed ID10545368
A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole, IANBD) ... More