Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments.
AuthorsDharmawardana KR, Olson ST, Bock PE
JournalJ Biol Chem
PubMed ID10373475
'The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled ... More
Kinetic studies of calcium binding to the regulatory site of troponin C from cardiac muscle.
AuthorsDong W, Rosenfeld SS, Wang CK, Gordon AM, Cheung HC
JournalJ Biol Chem
PubMed ID8557674
'We have studied the kinetics of the structural transitions induced by calcium binding to the single, regulatory site of cardiac troponin C by measuring the rates of calcium-mediated fluorescence changes with a monocysteine mutant of the protein (C35S) specifically labeled at Cys-84 with the fluorescent probe 2(-)[4''-(iodoacetamido)anilino]naphthalene-6-sulfonic acid. At 4 ... More
Inhibition of cathepsin L-like cysteine proteases by cytotoxic T-lymphocyte antigen-2 beta.
AuthorsDelaria K, Fiorentino L, Wallace L, Tamburini P, Brownell E, Muller D
JournalJ Biol Chem
PubMed ID7929206
'The protein sequence of cytotoxic T-lymphocyte antigen-2 beta (CTLA-2 beta) is 36% identical to the proregion of mouse cathepsin L (Denizot, F., Brunet, J.F., Roustan, P., Harper, K., Suzan, M., Luciani, M. F., Mattei, M. G., and Goldstein, P. (1989) Eur. J. Immunol. 19, 631-635). Here we report the expression, ... 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
Cysteine-reactive fluorescence probes of catalytic sites of ATP synthase.
'We searched for new fluorescent probes of catalytic-site nucleotide binding in F(1)F(0)-ATP synthase by introducing Cys mutations at positions in or close to catalytic sites and then reacting Cys-mutant F(1) with thiol-reactive fluorescent probes. Four suitable mutant/probe combinations were identified. beta F410C labeled by 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) gave very large signal ... More
Calcium-induced conformational change in cardiac troponin C studied by fluorescence probes attached to Cys-84.
AuthorsDong WJ, Cheung HC
JournalBiochim Biophys Acta
PubMed ID8695639
'Residue Cys-84 of bovine cardiac troponin C (cTnC) located at the C-terminal end of helix D was selectively labeled in the presence of Ca2+ with two fluorescent probes: IAANS (2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) and acrylodan (6-acrylol-2-(dimethylamino)naphthalene). The fluorescence of the attached probes was studied by the steady-state and time-resolved methods to gain ... More
Isoform specific interactions of troponin I and troponin C determine pH sensitivity of myofibrillar Ca2+ activation.
AuthorsBall KL, Johnson MD, Solaro RJ
JournalBiochemistry
PubMed ID8031779
'We investigated whether differences in isoforms of troponin I (TnI) and troponin C (TnC) can account for the greater inhibition of Ca(2+)-dependent MgATPase activity by acidic pH in cardiac (c) than in fast skeletal (fs) myofilaments. We studied fast skeletal myofibrils from which whole Tn was extracted by displacement with ... More
Fluorescence studies of red blood cell membranes from individuals with Huntington's disease.
AuthorsSumbilla C, Lakowicz JR
JournalJ Neurochem
PubMed ID6210762
'Fluorescence spectroscopic methods were used to investigate and compare the properties of erythrocyte membranes from individuals with Huntington''s Disease (HD) and from normal individuals. Erythrocyte ghosts were labeled with four different fluorescent probes: 1,6-diphenylhexatriene (DPH); 6-lauroyl-2-(dimethylamino)-naphthalene (Laurdan); 2-(4-maleimide anilino)-naphthalene-6-sulfonic acid (MIANS) and 5-(iodoacetamidoethyl)aminoaphthalene-1-sulfonic acid (IAEDANS). DPH is sensitive to the ... More
Phosphorylation-induced distance change in a cardiac muscle troponin I mutant.
AuthorsDong WJ, Chandra M, Xing J, She M, Solaro RJ, Cheung HC
JournalBiochemistry
PubMed ID9184157
'Phosphorylation of two adjacent serine residues in the unique N-terminal extension of cardiac muscle troponin I (cTnI) is known to decrease the Ca2+-sensitivity of cardiac myofilaments. To probe the structural significance of the N-terminal extension, we have constructed two cTnI mutants each containing a single cysteine: (1) a full-length cTnI ... 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
A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle.
AuthorsDong WJ, Wang CK, Gordon AM, Rosenfeld SS, Cheung HC
JournalJ Biol Chem
PubMed ID9235915
'The kinetics of the binding of Ca2+ to the single regulatory site of cardiac muscle troponin was investigated by using troponin reconstituted from the three subunits, using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe 2-[(4'-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-35. The kinetic tracings of binding experiments ... More
Structural consequences of cardiac troponin I phosphorylation.
AuthorsWard DG, Cornes MP, Trayer IP,
JournalJ Biol Chem
PubMed ID12207022
'beta-Adrenergic stimulation of the heart results in bisphosphorylation of the N-terminal extension of cardiac troponin I (TnI). Bisphosphorylation of TnI reduces the affinity of the regulatory site on troponin C (TnC) for Ca(2+) by increasing the rate of Ca(2+) dissociation. What remains unclear is how the phosphorylation signal is transmitted ... 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
Regulatory properties of recombinant tropomyosins containing 5-hydroxytryptophan: Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site.
AuthorsFarah CS, Reinach FC
JournalBiochemistry
PubMed ID10441151
'We have introduced tryptophan codons at different positions of the chicken alpha-tropomyosin cDNA (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466) and employed a trp auxotrophic Escherichia coli strain to express the proteins in media containing either normal tryptophan, ... More
Creatine kinase: a review of some recent work on the mechanism and subunit behaviour of the enzyme.
AuthorsBickerstaff GF, Price NC
JournalInt J Biochem
PubMed ID344081
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
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
Structure-function analysis of HscC, the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones.
'Hsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release. In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E. coli ... More
Effects of temperature and reagent size on the reaction of the thiol groups of rabbit muscle creatine kinase [proceedings]
AuthorsPrice NC
JournalBiochem Soc Trans
PubMed ID902911
Myosin structure. Proximity measurements by fluorescence energy transfer.
AuthorsHaugland RP
JournalJ Supramol Struct
PubMed ID127888
Time-resolved fluorescence studies on the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.
AuthorsHanemaaijer R, Masurel R, Visser AJ, de Kok A, Veeger C
JournalFEBS Lett
PubMed ID3169263
The dihydrolipoyl transacetylase (E2) component of A. vinelandii PDC and its lipoyl domain shows similar dynamic properties as revealed with fluorescence anisotropy decay of lipoyl-bound IAANS. The lipoyl domain (32.6 kDa), containing three almost identical subdomains shows a mode of rotation characteristic for a protein of about 30 kDa. A ... More
Equilibrium titration of protein-ligand binding affinity in the presence of volatile reagents--a semiautomated approach.
AuthorsBreukelmann D, Housmans PR
JournalAnal Biochem
PubMed ID12576062
An existing method of equilibrium titration was significantly improved for investigating the effects of volatile anesthetics on Ca(2+) binding characteristics of human recombinant cardiac troponin C in in vitro conditions. The modified method increases stability of volatile compound concentrations in solution and allows for faster and more accurate data acquisition. ... More
The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding.
AuthorsPrice NC, Stevens E
JournalBiochem J
PubMed ID6805463
A number of aspects of the refolding of denatured rabbit muscle creatine kinase have been studied. Addition of substrates has no effect on the rate or extent of regain of activity. The changes in protein fluorescence during refolding broadly parallel the regain of activity. A study of the susceptibility of ... 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
A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus.
AuthorsD'Auria S, DiCesare N, Staiano M, Gryczynski Z, Rossi M, Lakowicz JR
JournalAnal Biochem
PubMed ID11950213
We describe the use of a thermostable glucokinase in a novel competitive fluorescence assay for glucose. Glucokinase from Bacillus stearothermophilus (BSGK) was found to retain enzymatic activity in solution for over 20 days. The single cysteine residue in BSGK, which is near the active site, was labeled with a fluorescent ... More
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
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
N,N'-dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofurazan bind to different beta subunits of the F1 ATPase of Escherichia coli.
AuthorsStan-Lotter H, Bragg PD
JournalArch Biochem Biophys
PubMed ID2873791
The fluorescent thiol reagent 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS) labels the gamma, delta, and one of the three beta subunits of the F1 ATPase from Escherichia coli (ECF1). This is the same beta subunit which incorporates 4-chloro-7-nitrobenzofurazan (Nbf) [H. Stan-Lotter and P. D. Bragg (1986) Eur. J. Biochem. 154, 321-327]. After inactivation ... More
Sulfhydryl modification of E. coli Cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex.
AuthorsMendoza JA, Horowitz PM
JournalJ Protein Chem
PubMed ID1361328
Differential chemical modification of E. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4'-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were ... More
Resonance energy transfer between the active sites of creatine kinase from rabbit brain.
AuthorsGrossman SH
JournalBiochim Biophys Acta
PubMed ID2400776
Resonance energy transfer was measured between the active site domains of the brain isozyme of creatine kinase (CK-BB). The reactive thiol near the active sites, one on each subunit of the dimeric protein, was derivatized using 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (AED), 2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid (AANS) and 5-iodoacetamidofluorescein (AF). Suitable donor/acceptor protein conjugated hybrids ... More
Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction.
AuthorsKnetsch ML, Uyeda TQ, Manstein DJ
JournalJ Biol Chem
PubMed ID10400626
The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between ... More
The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide.
AuthorsPrice NC
JournalBiochem J
PubMed ID435254
The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, ... More
Regulatory domain conformational exchange and linker region flexibility in cardiac troponin C bound to cardiac troponin I.
AuthorsAbbott MB, Gaponenko V, Abusamhadneh E, Finley N, Li G, Dvoretsky A, Rance M, Solaro RJ, Rosevear PR
JournalJ Biol Chem
PubMed ID10801883
Previously, we utilized (15)N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca(2+)-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I (Gaponenko, V., Abusamhadneh, E., Abbott, M. B., Finley, N., Gasmi-Seabrook, G., Solaro, R.J., Rance, ... More
Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation.
AuthorsGuo X, Wattanapermpool J, Palmiter KA, Murphy AM, Solaro RJ
JournalJ Biol Chem
PubMed ID8195157
Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. ... More
Functional consequences of troponin T mutations found in hypertrophic cardiomyopathy.
AuthorsTobacman LS, Lin D, Butters C, Landis C, Back N, Pavlov D, Homsher E
JournalJ Biol Chem
PubMed ID10497196
Missense mutations in the cardiac thin filament protein troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). To understand how these mutations produce dysfunction, five TnTs were produced and purified containing FHC mutations found in several regions of TnT. Functional defects were diverse. Mutations F110I, E244D, and COOH-terminal ... More
Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin.
AuthorsLiaw PC, Becker DL, Stafford AR, Fredenburgh JC, Weitz JI
JournalJ Biol Chem
PubMed ID11294849
Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin ... More
A fluorescent probe study of Ca2+ binding to the Ca2+-specific sites of cardiac troponin and troponin C.
AuthorsJohnson JD, Collins JH, Robertson SP, Potter JD
JournalJ Biol Chem
PubMed ID7430090
Cardiac troponin C (C-TnC) was labeled with the sulfhydryl-specific fluorescent probe molecule 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid at cysteine 35 and 84 to produce C-TnCIA. This modified protein binds Ca2+, undergoes Ca2+-induced increases in alpha helix, and forms a complex with other troponin subunits as does unlabeled C-TnC. C-TnCIA undergoes a small fluorescence ... 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
Coupling of calcium to the interaction of troponin I with troponin C from cardiac muscle.
AuthorsLiao R, Wang CK, Cheung HC
JournalBiochemistry
PubMed ID7918499
The interaction of troponin I (CTnI) with troponin C (CTnC) from bovine cardiac muscle was studied using CTnC modified at Cys35 and Cys84 with the fluorescent probe 2-[(4'-iodoacetamido)-anilino]naphthalene-6-sulfonic acid (CTnCIAANS). The association constant for complex formation between the two proteins was determined at 20 degrees C in 0.4 M KCl, ... More
Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen.
AuthorsBoxrud PD, Fay WP, Bock PE
JournalJ Biol Chem
PubMed ID10799544
Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and ... More
Binding of fibrin monomer and heparin to thrombin in a ternary complex alters the environment of the thrombin catalytic site, reduces affinity for hirudin, and inhibits cleavage of fibrinogen.
AuthorsHogg PJ, Jackson CM, Labanowski JK, Bock PE
JournalJ Biol Chem
PubMed ID8824251
Interaction of the blood clotting proteinase, thrombin, with fibrin monomer and heparin to form a thrombin.fibrin monomer.heparin ternary complex is accompanied by a change in thrombin catalytic specificity. Equilibrium binding interactions in the assembly of the ternary complex were characterized quantitatively using thrombin labeled at the active site with a ... More
Alteration around the active site of rhodanese during urea-induced denaturation and its implications for folding.
AuthorsBhattacharyya AM, Horowitz P
JournalJ Biol Chem
PubMed ID10809729
The enzyme rhodanese contains two globular domains connected by a tether region and associated by strong hydrophobic interactions. The protein has proven to be very difficult to refold without assistance to prevent oxidation and aggregation. For this study, the active site cysteine 247, near the interdomain region, was modified with ... More
Fluorescent adducts of wheat calmodulin implicate the amino-terminal region in the activation of skeletal muscle myosin light chain kinase.
AuthorsZot HG, Aden R, Samy S, Puett D
JournalJ Biol Chem
PubMed ID2394697
Considerable attention is being directed toward defining a binding site in the central region of calmodulin that forms a high affinity interaction with certain enzymes and amphiphilic peptides. However, other regions of calmodulin are also known to be involved in the activation of enzymes such as myosin light chain kinase, ... More
Fast skeletal muscle skinned fibers and myofibrils reconstituted with N-terminal fluorescent analogues of troponin C.
AuthorsZot HG, Güth K, Potter JD
JournalJ Biol Chem
PubMed ID2946678
Glycerinated rabbit fast skeletal muscle fibers were chemically skinned with 1% Brij 35 and partially depleted of endogenous troponin C subunit (TnC) by exposure of the fibers to EDTA (Zot, H. G., and Potter, J. D. (1982) J. Biol. Chem. 257, 7678-7683). The TnC-depleted fibers exhibited a decrease in maximal ... More
Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment.
AuthorsStrandberg L, Karolin J, Johansson LB, Fa M, Aleshkov S, Ny T
JournalThromb Res
PubMed ID7863476
To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to ... More
The effect of pH on the Ca2+ affinity of the Ca2+ regulatory sites of skeletal and cardiac troponin C in skinned muscle fibres.
AuthorsParsons B, Szczesna D, Zhao J, Van Slooten G, Kerrick WG, Putkey JA, Potter JD
JournalJ Muscle Res Cell Motil
PubMed ID9350012
It is known that intracellular pH drops rapidly after the onset of ischemia in cardiac muscle and may play some role in the rapid drop in force that ensues. It is also known that alpha 1-adrenoceptor agonists alkalinize intracellular pH by stimulating Na+/H+ exchange and may represent a mechanism which ... 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
The kinetic cycle of cardiac troponin C: calcium binding and dissociation at site II trigger slow conformational rearrangements.
The goal of this study is to characterize the kinetic mechanism of Ca2+ activation and inactivation of cardiac troponin C (cTnC), the Ca2+ signaling protein which triggers heart muscle contraction. Previous studies have shown that IAANS covalently coupled to Cys84 of wild-type cTnC is sensitive to conformational change caused by ... More
Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases.
AuthorsBock PE
JournalJ Biol Chem
PubMed ID1634536
The behavior of an array of fluorescent human alpha-thrombin derivatives in reporting binding of the fragment 2 domain of prothrombin was characterized as a representative application of the active-site-selective labeling approach to studies of blood coagulation proteinase regulatory interactions. An array of 16 thrombin derivatives was prepared by affinity labeling ... More
The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation.
AuthorsYe J, Esmon NL, Esmon CT, Johnson AE
JournalJ Biol Chem
PubMed ID1660464
The association of thrombin with thrombomodulin, a non-enzymatic endothelial cell surface receptor, alters the substrate specificity of thrombin. Complex formation converts thrombin from a procoagulant to an anticoagulant enzyme. Structure-function analysis of this change in specificity is facilitated by the availability of two soluble proteolytic derivatives of thrombomodulin, one consisting ... More
The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.
AuthorsYe J, Liu LW, Esmon CT, Johnson AE
JournalJ Biol Chem
PubMed ID1317850
The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing ... More
The chondroitin sulfate moiety of thrombomodulin binds a second molecule of thrombin.
AuthorsYe J, Esmon CT, Johnson AE
JournalJ Biol Chem
PubMed ID8381406
The role of the chondroitin sulfate moiety of thrombomodulin (TM) in the binding of thrombin to TM has been examined using fluorescent derivatives of thrombin. An anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg (FPR) linkage to form ANS-FPR-thrombin. When ANS-FPR-thrombin ... More
Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.
AuthorsKleerekoper Q, Howarth JW, Guo X, Solaro RJ, Rosevear PR
JournalBiochemistry
PubMed ID7577919
Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys ... More
Analogs of human plasminogen that are labeled with fluorescence probes at the catalytic site of the zymogen. Preparation, characterization, and interaction with streptokinase.
AuthorsBock PE, Day DE, Verhamme IM, Bernardo MM, Olson ST, Shore JD
JournalJ Biol Chem
PubMed ID8557633
Fluorescent analogs of the proteinase zymogen, plasminogen (Pg), which are specifically inactivated and labeled at the catalytic site have been prepared and characterized as probes of the mechanisms of Pg activation. The active site induced non-proteolytically in Pg by streptokinase (SK) was inactivated stoichiometrically with the thioester peptide chloromethyl ketone. ... More
Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity.
AuthorsSolaro RJ, el-Saleh SC, Kentish JC
JournalMol Cell Biochem
PubMed ID2530435
When the pH surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca2+-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca2+-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH ... More
Ca2+ - and cross-bridge-dependent changes in N- and C-terminal structure of troponin C in rat cardiac muscle.
AuthorsMartyn DA, Regnier M, Xu D, Gordon AM
JournalBiophys J
PubMed ID11159408
Linear dichroism of 5'-tetramethylrhodamine (5'ATR)-labeled cardiac troponin C (cTnC) was measured to monitor cTnC structure during Ca2+-activation of force in rat skinned myocardium. Mono-cysteine mutants allowed labeling at Cys-84 (cTnC(C84), near the D/E helix linker); Cys-35 (cTnC(C35), at nonfunctional site I); or near the C-terminus with a cysteine inserted at ... More
Effects of protein kinase A phosphorylation on signaling between cardiac troponin I and the N-terminal domain of cardiac troponin C.
AuthorsChandra M, Dong WJ, Pan BS, Cheung HC, Solaro RJ
JournalBiochemistry
PubMed ID9341222
During beta-adrenergic stimulation of the heart, there is a decrease in myofilament Ca2+ sensitivity mediated by the protein kinase A-(PKA-) induced phosphorylation of troponin I (cTnI). Phosphorylation, which occurs at Ser 23 and Ser 24 in an amino-terminal extension unique to cTnI, decreases the Ca2+ affinity of the amino-terminal regulatory ... More
Static and kinetic studies on rabbit skeletal muscle troponin.
AuthorsNishio T, Iio T
JournalJ Biochem (Tokyo)
PubMed ID6643420
Fluorescence titration curves of 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled troponin (IAANS-labeled Tn) and troponin-1-anilinonaphthalene-8-sulfonic acid (Tn-ANS) complex indicated that the fluorescent moiety, IAANS or ANS, detects conformational change of troponin I (TnI) or Tn due to the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of troponin C (TnC) component. ... More
Fluorescent probes attached to Cys 35 or Cys 84 in cardiac troponin C are differentially sensitive to Ca(2+)-dependent events in vitro and in situ.
AuthorsPutkey JA, Liu W, Lin X, Ahmed S, Zhang M, Potter JD, Kerrick WG
JournalBiochemistry
PubMed ID9020797
The goal of the current study was to generate recombinant cTnC proteins with single Cys residues as sites for attachment of fluorescent probes that can distinguish between the structural effects of myosin cross bridges and direct Ca2+ binding to cTnC (cardiac and slow skeletal troponin C) in skinned fibers. We ... 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
Determination of affinities for lck SH2 binding peptides using a sensitive fluorescence assay: comparison between the pYEEIP and pYQPQP consensus sequences reveals context-dependent binding specificity.
AuthorsCousins-Wasti RC, Ingraham RH, Morelock MM, Grygon CA
JournalBiochemistry
PubMed ID8988011
The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the ... More
Disparate fluorescence properties of 2-[4'-(iodoacetamido)anilino]-naphthalene-6-sulfonic acid attached to Cys-84 and Cys-35 of troponin C in cardiac muscle troponin.
AuthorsDong WJ, Wang CK, Gordon AM, Cheung HC
JournalBiophys J
PubMed ID9017210
Two monocysteine mutants of cardiac muscle troponin C, cTnC(C35S) and cTnC(C84S), were genetically generated and labeled with the fluorescent probe 2-[4'-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-84 and Cys-35, respectively. Cys-84 is located on helix D in the regulatory N-domain, and Cys-35 is at the -y position of the inactive 12-residue loop ... More
The ELF-97 alkaline phosphatase substrate provides a bright, photostable, fluorescent signal amplification method for FISH.
We used the ELF-97 (Enzyme-Labeled Fluorescence) phosphatase substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at approximately 360 nm, with emission centered at approximately 530 ... More
Energy-transfer measurements of the Cys35-Cys84 distance in bovine cardiac troponin C.
AuthorsLiou YM, Fuchs F
JournalBiochim Biophys Acta
PubMed ID8373830
Bovine cardiac troponin C (cTnC) has cysteine residues located in the non-functional Ca(2+)-binding loop I (Cys-35) and at the N-terminal end of the central helix (Cys-84) near site II, the regulatory Ca(2+)-binding site. Recently, we reported that the excimer fluorescence resulting from the dimerization of adjacent pyrene groups attached to ... More
Conformation of the N-terminal segment of a monocysteine mutant of troponin I from cardiac muscle.
AuthorsDong WJ, Chandra M, Xing J, Solaro RJ, Cheung HC
JournalBiochemistry
PubMed ID9184156
A monocysteine mutant of cardiac muscle troponin I, cTnI(S5C/C81I/C98S), was generated from a mouse cTnI cDNA clone and expressed in a bacterial system. Cys-5 was modified with the fluorescent sulfhydryl reagent IAANS to probe the conformation of the N-terminal extension of the mutant and the mutant complexed with cardiac muscle ... More