Rotational diffusion of human lipoproteins and their receptors as determined by time-resolved phosphorescence anisotropy.
AuthorsTilley L, Sawyer WH, Morrison JR, Fidge NH
JournalJ Biol Chem
PubMed ID2846567
'Time-resolved phosphorescence anisotropy has been used to assess the rotational dynamics of human serum lipoproteins labeled with phosphorescent probes of high triplet yield. Labeling the lipid phase of low density, very low density, and high density lipoproteins with an eosinyl fatty acid revealed the existence of two motions. The shorter ... More
Interactions of troponin subunits: free energy of binary and ternary complexes.
AuthorsCheung HC, Wang CK, Malik NA
JournalBiochemistry
PubMed ID3676297
'We have determined the free energy of formation of the binary complexes formed between skeletal troponin C and troponin T (TnC.TnT) and between troponin T and troponin I (TnT.TnI). This was accomplished by using TnC fluorescently modified at Cys-98 with N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine for the first complex and TnI labeled at Cys-133 ... More
Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase.
AuthorsNigg E, Kessler M, Cherry RJ
JournalBiochim Biophys Acta
PubMed ID215229
'The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate ... More
Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer.
AuthorsWang YL, Taylor DL
JournalCell
PubMed ID6101197
'Fluorescence resonance energy transfer was used to follow the dynamic equilibrium of actin polymerization. We prepared fluorescent analogs of actin by labeling actin covalently with fluorescein (as the donor) and with eosin (as the acceptor). The copolymer of donor- and acceptor-labeled actin exhibits a 60%-70% efficiency of energy transfer. We ... More
Detection of actin assembly by fluorescence energy transfer.
AuthorsTaylor DL, Reidler J, Spudich JA, Stryer L
JournalJ Cell Biol
PubMed ID6894758
'Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor-labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole ... More
Binary interactions of troponin subunits.
AuthorsIngraham RH, Swenson CA
JournalJ Biol Chem
PubMed ID6204984
'The association constants for the formation of the binary complexes of rabbit fast skeletal muscle troponin subunits have been determined for three solution conditions: (a) 1 mM CaCl2, (b) 3 mM MgCl2 and 1 mM EGTA, and (c) 2 mM EDTA. The subunits were labeled with extrinsic fluorescence probes, either ... More
Self-quenched fluorogenic protein substrates for the detection of cathepsin D and other protease activities.
AuthorsAshcom JD, Jacobson LA
JournalAnal Biochem
PubMed ID2662807
'Self-quenched fluorogenic substrates for proteolytic enzymes have been prepared by alkylation of thiol groups in reduced bovine serum albumin with iodoacetamidofluorescein or iodoacetamidoeosin. Substrates immobilized by adsorption onto nitrocellulose membranes or by incorporation into agarose gel slabs are suitable for fluorescence zymography after electrophoretic separation of catalytically active proteases, including ... More
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study.
AuthorsBoskovic DS, Troxler T, Krishnaswamy S
JournalJ Biol Chem
PubMed ID14988397
'The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal ... More
Structural organization of the synaptic exocytosis core complex.
AuthorsLin RC, Scheller RH
JournalNeuron
PubMed ID9390521
'Syntaxin, vesicle-associated membrane protein (VAMP), and synaptosome-associated protein of 25 kDa (SNAP-25) form a ternary "core complex" central to the process of synaptic vesicle docking and fusion. Several lines of evidence support the hypothesis that the proteins assemble in a coiled-coil structure, but the alignment of alpha helices in this ... More
Rotational motion and flexibility of Ca2+,Mg2+-dependent adenosine 5'-triphosphatase in sarcoplasmic reticulum membranes.
AuthorsBürkli A, Cherry RJ
JournalBiochemistry
PubMed ID6162478
'Ca2+,Mg2+-dependent adenosine 5''-triphosphatase (ATPase) in sarcoplasmic reticulum vesicles is labeled with the triplet probe, 5-iodoacetamidoesin. Rotational mobility of the ATPase is investigated by measuring flash-induced transient dichroism of the eosin probe. The absorption anisotropy measured 20 mus after the exciting flash is found to be small at 37 degrees C ... More
Resonance energy transfer between catalytic sites of bovine liver uridine diphosphoglucose dehydrogenase.
AuthorsFranzen JS, Marchetti PS, Feingold DS
JournalBiochemistry
PubMed ID7470452
Fluorescence energy transfer studies of skeletal troponin C proximity between methionine-25 and cysteine-98.
AuthorsCheung HC, Wang CK, Garland F
JournalBiochemistry
PubMed ID7171543
Rotational and lateral diffusion of membrane proteins.
AuthorsCherry RJ
JournalBiochim Biophys Acta
PubMed ID391281
Internal motion of F-actin in 10(-6)-10(-3) s time range studied by transient absorption anisotropy: detection of torsional motion.
AuthorsMihashi K, Yoshimura H, Nishio T, Ikegami A, Kinosita K
JournalJ Biochem (Tokyo)
PubMed ID6885746
By means of laser flash photolysis, the transient absorption anisotropy (TAA) of the triplet probe, 5-iodoacetamide-Eosin, labeling rabbit skeletal F-actin was measured in the 10(-6)-10(-3) s time range. The TAA curve at 20 degrees C showed a relatively slow decay phase covering several hundred microseconds and a large residual anisotropy ... More
Detection of DNA bending in a DNA-PAP1 protein complex by fluorescence resonance energy transfer.
AuthorsOzaki H, Iwase N, Sawai H, Kodama T, Kyogoku Y
JournalBiochem Biophys Res Commun
PubMed ID9070843
The structure of DNA in a DNA-protein complex was studied by means of fluorescence resonance energy transfer (FRET) method. Oligonucleotide phosphorothioates were labeled with fluorescein and eosin to obtain a donor- and acceptor-labeled DNA. The formation of a complex of the DNA with PAP1(70), which is a DNA binding site ... More
Fluorescence energy transfer study of troponin C-melittin complex.
AuthorsSano H, Iio T
JournalJ Biochem (Tokyo)
PubMed ID8749318
Fluorescence energy transfer study of rabbit skeletal troponin C (TnC), which binds a donor, dansylaziridine, to Met-25 in helix A and an acceptor, 5-(iodoacetamido)eosin, to Cys-98 in helix E revealed (i) the donor-acceptor distance did not change upon Mg2+ and Ca2+ binding to TnC, (ii) melittin binding to Ca(2+)-free TnC ... More
Rotational molecular dynamics of the membrane-bound acetylcholine receptor revealed by phosphorescence spectroscopy.
AuthorsBartholdi M, Barrantes FJ, Jovin TM
JournalEur J Biochem
PubMed ID6172277
The rotational mobility of the membrane-bound acetylcholine receptor from Torpedo marmorata has been studied by phosphorescence anisotropy techniques using eosin-5'-isothiocyanate and eosin-5'-iodoacetamide derivatives of the alpha-neurotoxin of Bungarus multicinctus (alpha-bungarotoxin). Normal membranes, those depleted of nonreceptor peripheral peptides by alkaline treatment, as well as membranes subjected to various chemical modifications ... More
Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation.
Authorsdos Remedios CG, Miki M, Barden JA
JournalJ Muscle Res Cell Motil
PubMed ID3298315
The contractile proteins actin and myosin are of considerable biological interest. They are essential for muscle contraction and in eukaryotic cells they play a crucial role in most contractile phenomena. Over the years since the first fluorescence resonance energy transfer (FRET) paper appeared, an extensive body of literature has accumulated ... More
Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status.
AuthorsCaldwell CC, Hornyak SC, Pendleton E, Campbell D, Knowles AF
JournalArch Biochem Biophys
PubMed ID11368171
The major ectonucleoside triphosphate phosphohydrolase in the chicken gizzard smooth muscle membranes is an ecto-ATPase, an integral membrane glycoprotein belonging to the E-ATPase (or E-NTPDase) family. The gizzard ecto-ATPase is distinguished by its unusual kinetic properties, temperature dependence, and response to a variety of modulators. Compounds that promote oligomerization of ... More
Chemical modification and fluorescence labeling study of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum using iodoacetamide and its N-substituted derivatives.
AuthorsBaba A, Nakamura T, Kawakita M
JournalJ Biochem (Tokyo)
PubMed ID2950079
Sarcoplasmic reticulum membrane vesicles from rabbit skeletal muscle were treated with iodoacetamide (IAA) at pH 7.0 and 30 degrees C. At 1.0 mM IAA, 1 mol of IAA per mol of ATPase peptide was bound in 1 h. Under these conditions, IAA was attached specifically to the B-tryptic fragment portion ... More
Determination of the radial coordinate of Cys-374 in F-actin using fluorescence resonance energy transfer spectroscopy: effect of phalloidin on polymer assembly.
AuthorsMoens PD, Yee DJ, dos Remedios CG
JournalBiochemistry
PubMed ID7947715
In helically symmetric protein assemblies, fluorescence resonance energy transfer (FRET) spectroscopy can be used to determine the radial coordinates of fluorescent probes attached to specific amino acid side chains. This is done by separately labeling monomers with donor and acceptor probes, mixing them in different proportions, allowing the mixtures to ... More
Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy.
AuthorsBrown LJ, Klonis N, Sawyer WH, Fajer PG, Hambly BD
JournalBiochemistry
PubMed ID11444974
Inter- and intradomain flexibility of the myosin head was measured using phosphorescence anisotropy of selectively labeled parts of the molecule. Whole myosin and the myosin head, subfragment-1 (S1), were labeled with eosin-5-iodoacetamide on the catalytic domain (Cys 707) and on two sites on the regulatory domain (Cys 177 on the ... More
The estimation of distances between specific backbone-labeled sites in DNA using fluorescence resonance energy transfer.
AuthorsOzaki H, McLaughlin LW
JournalNucleic Acids Res
PubMed ID1408835
A series DNA helices of twenty-four base pairs has been prepared for the study of fluorescence resonance energy transfer. Each of the DNA helices contains two phosphorothioate diesters (one in each strand) at pre-selected sites for introduction of the desired donor and acceptor fluorophores. The phosphorothioate-containing oligodeoxynucleotides have been prepared ... More
Distance distributions and anisotropy decays of troponin C and its complex with troponin I.
AuthorsCheung HC, Wang CK, Gryczynski I, Wiczk W, Laczko G, Johnson ML, Lakowicz JR
JournalBiochemistry
PubMed ID2036391
We used frequency domain measurements of fluorescence resonance energy transfer to recover the distribution of distances between Met 25 and Cys 98 in rabbit skeletal troponin C. These residues were labeled with dansylaziridine as energy donor and 5-(iodoacetamido)eosin as acceptor and are located on the N- and C-terminal lobes of ... More
Regulatory and catalytic domain dynamics of smooth muscle myosin filaments.
AuthorsLi HC, Song L, Salzameda B, Cremo CR, Fajer PG
JournalBiochemistry
PubMed ID16681394
Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, tau(r), were 24 +/- 6 micros and 441 ... More
Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence.
AuthorsEads TM, Thomas DD, Austin RH
JournalJ Mol Biol
PubMed ID6209402
We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after ... More
Proximity relationship in the binary complex formed between troponin I and troponin C.
AuthorsWang CK, Cheung HC
JournalJ Mol Biol
PubMed ID2950237
We have determined six molecular distances among four sites in the binary complex formed between troponin C (TnC) and troponin I (TnI) by fluorescence resonance energy transfer between donor and acceptor probes that were either an intrinsic fluorophore (Trp158 of TnI) or extrinsic probes attached to the sites. The three ... More
Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.
AuthorsLoscalzo J
JournalJ Clin Invest
PubMed ID3139714
We examined the similarities and differences in conformation between recombinant human single-chain tissue plasminogen activator (sct-PA) and two-chain tissue plasminogen activator (tct-PA), and compared these structural data with measurement of enzymatic activity. The intrinsic protein fluorescence of native tct-PA was 54% that of sct-PA. Differences in steady state protein fluorescence ... More
Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.
AuthorsWang CK, Cheung HC
JournalBiophys J
PubMed ID4074834
We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled ... More