The influence of divalent cations on the dynamic properties of actin filaments: a spectroscopic study.
AuthorsHild G, Nyitrai M, Belágyi J, Somogyi B
JournalBiophys J
PubMed ID9826621
The principal aim of this investigation was to study the change of the protein flexibility and/or conformational properties of actin filaments upon the replacement of Ca2+ by Mg2+. The temperature dependence of the fluorescence lifetime and the anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) attached covalently to the Cys374 residue of actin ... More
Sulfhydryl groups of rabbit liver arylsulfatase A.
AuthorsWaheed A, Van Etten RL
JournalBiochim Biophys Acta
PubMed ID2864083
Rabbit liver arylsulfatase A (aryl-sulfate sulfhydrolase, EC 3.1.6.1) monomers of 130 kDa contain two free sulfhydryl groups as determined by spectrophotometric titration using 5,5'-dithiobis(2-nitrobenzoate) and by labeling with the fluorescent probe 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Fluorescence quenching data indicate that the reactive sulfhydryl is present in proximity to one or more tryptophan ... More
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
Time-resolved fluorescence depolarization measurements of F-actin binding to myosin subfragments-1 bearing different alkali light chains.
AuthorsWadzinski L, Botts J, Wang A, Woodward J, Highsmith S
JournalArch Biochem Biophys
PubMed ID42352
Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy.
AuthorsLim K, Jameson DM, Gentry CA, Herron JN
JournalBiochemistry
PubMed ID7766607
'Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization ... More
Fluorescence studies on the interaction of muscle M-line proteins, creatine kinase and the 165,000 dalton component, with each other and with myosin and myosin subfragments.
AuthorsMani RS, Kay CM
JournalInt J Biochem
PubMed ID7319124
Derivatization strategies for preparing N-glycan probes.
AuthorsRice KG
JournalAnal Biochem
PubMed ID10929802
Involvement of conserved, acidic residues in the N-terminal domain of troponin C in calcium-dependent regulation.
AuthorsKobayashi T, Zhao X, Wade R, Collins JH
JournalBiochemistry
PubMed ID10220325
'We have mutated eight conserved, charged amino acid residues in the N-terminal, regulatory domain of troponin C (TnC) so we could investigate their role in troponin-linked Ca2+ regulation of muscle contraction. These residues surround a hydrophobic pocket in the N-terminal domain of TnC which, when Ca2+ binds to regulatory sites ... More
Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin.
AuthorsGraceffa P
JournalBiochemistry
PubMed ID9092808
'Smooth muscle caldesmon is a single polypeptide chain with its NH2- and COOH-terminal domains separated by a long alpha-helix. Caldesmon was labeled at either Cys-153 in the NH2 domain or Cys-580 in the COOH domain with a variety of fluorescence probes. Fluorescence intensity, peak position, and polarization of probes on ... More
The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle.
AuthorsChen B, Jones TE, Bigelow DJ
JournalBiochemistry
PubMed ID10555971
'Cellular conditions in senescent skeletal muscle have been shown to result in the loss of conformational stability of the sarcoplasmic reticulum (SR) Ca-ATPase. To identify underlying structural features of age-modified Ca-ATPase, we have utilized the fluorescence properties of protein-bound probes to assess both local and global structure. We find conformational ... More
Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses.
AuthorsTakai Y, Mizuochi T, Fujiwara H, Hamaoka T
JournalJ Immunol
PubMed ID6197460
'Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study ... More
The binding of myosin subfragment-1 to F-actin in the absence of nucleotide. Evidence for dependence on F-actin concentration.
AuthorsYoshimura H, Mihashi K
JournalJ Biochem (Tokyo)
PubMed ID7130152
'The binding of myosin subfragment-1 (S-1) to F-actin in the absence of nucleotide was examined by the sedimentation method using 1,5-IAEDANS-labeled S-1. We found that the binding affinity of F-actin to S-1 was dependent on the concentration of F-actin, and the binding was weaker at higher concentrations of F-actin. The ... More
Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative.
AuthorsYamashita T, Kawakita M
JournalJ Biochem (Tokyo)
PubMed ID2953712
'Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of ... More
Differential behavior of two cysteine residues on the myosin head in muscle fibers.
AuthorsMiyanishi T, Borejdo J
JournalBiochemistry
PubMed ID2523734
'We have previously shown that the orientation of (iodoacetamido)tetramethylrhodamine labels on SH1 thiol of S-1 moieties changes when MgADP is added to the fibers in rigor [Borejdo, J., Assulin, O., Ando, T., & Putnam, S. (1982) J. Mol. Biol. 158, 391-414. Burghardt, T.P., Ando, T., & Borejdo, J. (1983) Proc. ... More
Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin.
AuthorsAguirre R, Gonsoulin F, Cheung HC
JournalBiochemistry
PubMed ID3801396
'Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], ... More
Spatial relationship between heads of dimeric Ncd in the presence of nucleotides and microtubules.
AuthorsHajdo L, Skowronek K, Kasprzak AA
JournalArch Biochem Biophys
PubMed ID14984201
'Kinesins are molecular motors that produce mechanical work at the expense of ATP hydrolysis. Here, we studied Ncd (non-claret disjunctional), a (-)-end-directed member of this superfamily. To gain insight into the mechanism by which Ncd generates force and movement, we measured distances between the heads in dimeric Ncd-250-700 using fluorescence ... More
Streptolysin O: inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation.
AuthorsAbdel Ghani EM, Weis S, Walev I, Kehoe M, Bhakdi S, Palmer M
JournalBiochemistry
PubMed ID10563803
'Streptolysin O is a four-domain protein toxin that permeabilizes animal cell membranes. The toxin first binds as a monomer to membrane cholesterol and subsequently assembles into oligomeric transmembrane pores. Binding is mediated by a C-terminally located tryptophan-rich motif. In a previous study, conformational effects of membrane binding were characterized by ... More
Properties of spin and fluorescent labels at a receptor-ligand interface.
AuthorsOwenius R, Osterlund M, Lindgren M, Svensson M, Olsen OH, Persson E, Freskgård PO, Carlsson U
JournalBiophys J
PubMed ID10512843
'Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently ... More
The flexibility of actin filaments as revealed by fluorescence resonance energy transfer. The influence of divalent cations.
AuthorsNyitrai M, Hild G, Belágyi J, Somogyi B
JournalJ Biol Chem
PubMed ID10224049
'The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f'', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility ... More
Conformation states of Xenopus transcription factor IIIA.
AuthorsHanas JS, Duke AL, Gaskins CJ
JournalBiochemistry
PubMed ID2502179
'The conformation of Xenopus transcription factor IIIA (TFIIIA) free in solution, bound to 5S RNA in the 7S particle, depleted of zinc, or bound to plasmid DNA was analyzed by (1) trypsin digestion and electrophoretic analysis of proteolytic fragments or (2) measurement of the fluorescence of TFIIIA mildly derivatized with ... More
Excitation energy transfer studies on the proximity between SH1 and the adenosinetriphosphatase site in myosin subfragment 1.
AuthorsTao T, Lamkin M
JournalBiochemistry
PubMed ID6457630
'Excitation energy transfer studies were carried out to determine the distance between the adenosinetriphosphatase (ATPase) site and a unique "fast-reacting" sulfhydryl (referred to as SH1) in myosin subfragment 1. The fluorescent moiety of the probe N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylene-diamine was used as the donor attached at SH1. The chromophoric nucleotide analogue 2''(3'')-0-(2,4,6-trinitrophenyl)adenosine 5''-diphosphate ... More
Soluble amyloid Abeta-(1-40) exists as a stable dimer at low concentrations.
AuthorsGarzon-Rodriguez W, Sepulveda-Becerra M, Milton S, Glabe CG
JournalJ Biol Chem
PubMed ID9261105
'Recent studies have implicated the amyloid Abeta peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble Abeta or its oligomeric state, and the existing data are often contradictory. In this study, we used intrinsic fluorescence ... More
Effect of salts on the stability and folding of staphylococcal nuclease.
AuthorsNishimura C, Uversky VN, Fink AL
JournalBiochemistry
PubMed ID11329280
'The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free ... More
Fluorescent labeling of the nucleotide site in cytosolic rat liver phosphoenolpyruvate carboxykinase.
AuthorsRojas MC, Encinas MV, Cardemil E
JournalArch Biochem Biophys
PubMed ID1897968
'Reaction of rat liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) with the alkylating fluorescent probe N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS), results in complete loss of enzymatic activity. One mole of the fluorescent reagent is incorporated per mole of the inactivated enzyme. When the modification is carried out in the presence ... More
Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation.
AuthorsBorejdo J, Putnam S
JournalBiochim Biophys Acta
PubMed ID849438
'Single skinned glycerinated muscle fibers were labelled with the fluorescent dye N-(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS). The heavy chain of myosin (EC 3.6.1.3) was labelled predominantly when the reaction was carried out in relaxation at 0 degrees C. Mechanical properties of skinned fibers were little affected by labelling with the fluorophore. Rigor ... More
Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products.
AuthorsChen T, Applegate D, Reisler E
JournalBiochemistry
PubMed ID3846455
'The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In ... More
Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin.
AuthorsCarlier MF, Pantaloni D, Korn ED
JournalJ Biol Chem
PubMed ID3814248
'The binding of cations to ATP-G-actin has been assessed by measuring the kinetics of the increase in fluorescence of N-acetyl-N''-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Ca2+ and Mg2+ compete for a single high-affinity site on ATP-G-actin with KD values of 1.5-15 nM for Ca2+ and 0.1-1 microM for Mg2+, i.e. with affinities 3-4 orders ... More
A phosphorylation-induced major structural change in the N-terminal domain of the P protein of Chandipura virus.
AuthorsRaha T, Chattopadhyay D, Chattopadhyay D, Roy S
JournalBiochemistry
PubMed ID10026294
'It has previously been shown that phosphorylation of P protein of vesicular stomatitis virus as well as Chandipura (CHP) virus is required for transcription activation and replication switch. The structural nature of this crucial conformational change, however, is largely unknown. We have studied the phosphorylation-associated conformational change in the P ... More
Structural connectivity in actin: effect of C-terminal modifications on the properties of actin.
AuthorsCrosbie RH, Miller C, Cheung P, Goodnight T, Muhlrad A, Reisler E
JournalBiophys J
PubMed ID7858132
'In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in ... More
Fluorescent modification of the cysteine 202 residue of Escherichia coli transcription termination factor rho.
AuthorsSeifried SE, Wang Y, von Hippel PH
JournalJ Biol Chem
PubMed ID2458348
'The lone cysteine residue (Cys-202) of transcription termination factor rho has been modified with the sulfhydryl-specific dyes 5-iodoacetamidofluorescein and 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid. Labeling with both dyes is specific for the Cys-202 residue and is at least 90% complete. Rho protein is an RNA-dependent ATPase and exists as a hexamer of identical ... More
Binding of heavy meromyosin and subfragment-1 to thin filaments in myofibrils and single muscle fibers.
AuthorsBorejdo J, Assulin O
JournalBiochemistry
PubMed ID7000187
'The binding of fluorescently labeled heavy meromyosin (HMM) and heavy meromyosin subfragment-1 (S-1) to thin filaments of myofibrils and of rabbit psoas muscle fibers was measured under conditions of rigor and contraction. The fragments diffused rapidly into the myofibrillar space and bound specifically to the thin filaments. The fragments bound ... More
Relocation of Cys374 of actin induced by labeling with fluorescent dyes.
AuthorsYasunaga T, Wakabayashi T
JournalJ Biochem (Tokyo)
PubMed ID11173519
'Cys374 is an important landmark of actin, because its sulfhydryl group is reactive and can be labeled with various reagents. The atomic coordinates of actin Cys374 have been determined by X-ray crystallography of co-crystals of actin with either profilin or gelsolin. However, the positions of Cys374 in the crystals determined ... More
Interhead fluorescence energy transfer between probes attached to translationally equivalent sites on the regulatory light chains of scallop myosin.
AuthorsChantler PD, Tao T
JournalJ Mol Biol
PubMed ID3820308
'Interhead fluorescence energy transfer studies between probes located at translationally equivalent sites on the two heads of scallop myosin indicates that the distance between such sites is no less than 50 A. Regulatory light chains, possessing either one (Mercenaria, chicken gizzard) or two (Loligo, rabbit skeletal) sulfhydryl groups, were modified ... More
Conformational changes in the vicinity of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle.
AuthorsObara M, Suzuki H, Kanazawa T
JournalJ Biol Chem
PubMed ID2964442
'In the previous experiment (Suzuki, H., Obara, M., Kuwayama, H., and Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456), the Ca2+-ATPase of sarcoplasmic reticulum vesicles was labeled with N-iodoacetyl-N''-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The main labeled site was Cys674. A large monophasic fluorescence drop occurred upon ATP ... More
Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate.
AuthorsShrestha S, Salins LL, Mark Ensor C, Daunert S
JournalBiotechnol Bioeng
PubMed ID12115121
'Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the ... 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 activation of the Ca-ATPase enhances conformational heterogeneity between nucleotide binding and phosphorylation domains.
AuthorsChen B, Squier TC, Bigelow DJ
JournalBiochemistry
PubMed ID15065881
'High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 degrees involving the nucleotide-binding (N) domain is required to permit the transfer of the gamma-phosphoryl group of ATP to Asp(351) in the phosphorylation (P) domain during coupled calcium transport. However, ... More
Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins.
AuthorsPutilina T, Skouri-Panet F, Prat K, Lubsen NH, Tardieu A
JournalJ Biol Chem
PubMed ID12562766
'The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for ... 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
Interaction of maleimidobenzoyl actin with myosin subfragment 1 and tropomyosin-troponin.
AuthorsMiki M, Hozumi T
JournalBiochemistry
PubMed ID1827994
'A chemical modification of G-actin with (m-maleimidobenzoyl)-N-hydroxysuccinimide ester (MBS) impairs actin polymerization [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. MBS-actin recovers the ability to polymerize when a 2-fold molar excess of phalloidin is added in 30 mM KCl/2 mM MgCl2/20 mM Tris-HCl ... More
Relaxation time, interthiol distance, and mechanism of action of ribosomal protein S1.
AuthorsOdom OW, Deng HY, Subramanian AR, Hardesty B
JournalArch Biochem Biophys
PubMed ID6201138
'The two sulfhydryl groups of ribosomal protein S1 from Escherichia coli have been labeled with fluorescent maleimides and the distance between them has been determined by nonradiative energy transfer. This distance was found to be approximately 27 A for both free S1 and S1 bound to 30 S subunits. This ... More
Intermediate conformational states of apocytochrome c.
AuthorsHamada D, Hoshino M, Kataoka M, Fink AL, Goto Y
JournalBiochemistry
PubMed ID8399178
'Horse apocytochrome c has been assumed to be a typical unfolded protein. At low ionic strength, the far- and near-UV circular dichroism spectra are typical of an unfolded protein at all pH values between 2 and 9. On the other hand, in the presence of high concentrations of salt, substantial ... More
Structural implications of the chemical modification of Cys(10) on actin.
AuthorsEli-Berchoer L, Reisler E, Muhlrad A
JournalBiophys J
PubMed ID10692333
'Cys(10) is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys(10) was modified with fluorescence probes N-(iodoacetyl)N''-(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4''-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromo bimane (MBB) by the method of, J. Biol. Chem. 266:5508-5513). The specificity of Cys(10) ... More
Interactions of the actin and nucleotide binding sites on myosin subfragment 1.
AuthorsHighsmith S
JournalJ Biol Chem
PubMed ID185204
'The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K''A, KN, and K''N in the scheme (see article), were determined for the magnesium ... More
Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin.
AuthorsRosenfeld SS, Xing J, Cheung HC, Brown F, Kar S, Sweeney HL
JournalJ Biol Chem
PubMed ID9786863
'In this study, we have examined the mechanism of phosphorylation-dependent regulation in smooth muscle myosin through the use of structural and kinetic methodologies applied to several myosin fragments. Fluorescence anisotropy decay measurements demonstrate that regulatory light chain phosphorylation significantly reduces the rotational correlation time of regulatable myosin preparations, whereas minimally ... More
Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates.
AuthorsTon-That H, Mazmanian SK, Faull KF, Schneewind O
JournalJ Biol Chem
PubMed ID10734144
'Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing ... More
Energy-transfer measurements on a double fluorescent labeled ribonuclease A.
AuthorsJullien M, Garel JR
JournalBiochemistry
PubMed ID6412748
'Two fluorescent groups have been covalently attached to ribonuclease A: first, the alpha-amino group is labeled upon reaction with fluorescein isothiocyanate, and second, one of the active site histidine residues is modified by N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid. Among the products of these two successive chemical modifications, a derivative bearing one label on ... More
Tropomyosin-troponin regulation of actin does not involve subdomain 2 motions.
AuthorsGerson JH, Kim E, Muhlrad A, Reisler E
JournalJ Biol Chem
PubMed ID11278830
'Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes ... More
Librational modes in liganded and unliganded hemoglobin as seen by fluorescence spectroscopy.
AuthorsSassaroli M, Bucci E, Steiner RF
JournalJ Biol Chem
PubMed ID7107598
'Hemoglobin labeled with N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate at the beta-93 cysteine shows normal allosteric properties including the Bohr effect and heme-heme-interaction, and the oxygen affinity is somewhat increased. Viscosity-resolved correlation times obtained from direct measurement of the decay of fluorescence anisotropy show values of 4.15 +/- 0.23 and 9.13 +/- 0.46 ns for ... More
Site-specific mutations in the myosin binding sites of actin affect structural transitions that control myosin binding.
AuthorsProchniewicz E, Thomas DD
JournalBiochemistry
PubMed ID11705383
'We have examined the effects of actin mutations on myosin binding, detected by cosedimentation, and actin structural dynamics, detected by spectroscopic probes. Specific mutations were chosen that have been shown to affect the functional interactions of actin and myosin, two mutations (4Ac and E99A/E100A) in the proposed region of weak ... More
tRNAfMet-induced conformational transition at the intersubunit domain of fluorescent-labeled methionyl-tRNA synthetase.
AuthorsFerguson BQ, Yang DC
JournalBiochemistry
PubMed ID3636154
'Conformational transition in methionyl-tRNA synthetase upon binding of tRNAfMet, whose binding shows strong negative cooporativity, was analyzed by fluorescence spectroscopy. The fluorescent probe N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS) reacts with native methionyl-tRNA synthetase in a nearly stoichiometric amount (2 per dimer) without affecting enzyme activity. The probe is shown by controlled trypsinization ... More
Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12.
'Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, ... 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
Low affinity interactions of GDPbetaS and ribose- or phosphoryl-substituted GTP analogues with the heterotrimeric G protein, transducin.
'We have examined the effects of three commonly used classes of guanine nucleotide analogues on the retinal G protein, transducin (Gt), and found them to be quite different from those that might be expected from results with other GTP-binding proteins. The most surprising results were with guanosine 5''-O-(2-thiodiphosphate) (GDPbetaS); rather ... More
The reduction of disulphides by rat liver mitochondria.
AuthorsJocelyn PC, Cronshaw AD
JournalBiochim Biophys Acta
PubMed ID6421329
'The capacity of rat liver mitochondria to reduce 23 non-protein disulphides to their thiols has been examined. The best reduced include the three intermolecular disulphides, bis(2-aminoethyl)disulphide (cystamine, basic), bis(2-hydroxyethyl)disulphide (HED, neutral and bis(3-carboxypropyl)disulphide (CPD, acidic). Their behaviour has been compared. In each case the thiol formed is found in highest ... More
Molecular dynamics of hemoglobin subunits as seen by fluorescence spectroscopy.
AuthorsOton J, Bucci E, Steiner RF, Fronticelli C, Franchi D, Montemarano J, Martinez A
JournalJ Biol Chem
PubMed ID7251596
'Fluorescent conjugates of beta A subunits and their respective heme-free derivatives have been prepared in which a 1,5-N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonate probe has been specifically placed at the beta-93 or beta-112 cysteine. The fluorescence anisotropy decay and static fluorescence polarization of these conjugates have been examined. Fluorescence measurements have also been made using ... More
The rates of switching movement of troponin T between three states of skeletal muscle thin filaments determined by fluorescence resonance energy transfer.
AuthorsShitaka Y, Kimura C, Miki M
JournalJ Biol Chem
PubMed ID15548522
'Troponin (Tn) plays the key roles in the regulation of striated muscle contraction. Tn consists of three subunits (TnT, TnC, and TnI). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the ... More
Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase.
'Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior ... More
Synthetic interface peptides as inactivators of multimeric enzymes: inhibitory and conformational properties of three fragments from Lactobacillus casei thymidylate synthase.
AuthorsPrasanna V, Bhattacharjya S, Balaram P
JournalBiochemistry
PubMed ID9578575
'Three synthetic peptides corresponding to distinct segments of the subunit interface of the dimeric enzyme thymidylate synthase (residues 17-38, N 22; residues 174-190, M 17; and residues 201-220, C 20) have been investigated for their ability to function as inhibitors by modifying the quaternary structure of the enzyme. A dramatic ... More
Minimalist aminoacylated RNAs as efficient substrates for elongation factor Tu.
AuthorsRudinger J, Blechschmidt B, Ribeiro S, Sprinzl M
JournalBiochemistry
PubMed ID8180193
'We demonstrate here, using RNA variants derived from tRNAAsp, that the minimalist aminoacylated structure able to interact efficiently with elongation factor Tu comprises a 10 base-pair helix linked to the 3''-terminal NCCA sequence. Shorter structures can interact with the elongation factor, but with significantly decreased affinity. Conserved features in the ... 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
High hydrostatic pressure perturbs the interactions between CF(0)F(1) subunits and induces a dual effect on activity.
AuthorsSouza MO, Creczynski-Pasa TB, Scofano HM, Gräber P, Mignaco JA
JournalInt J Biochem Cell Biol
PubMed ID15006644
'Chloroplast ATP-synthase is an H(+)/ATP-driven rotary motor in which a hydrophobic multi-subunit assemblage rotates within a hydrophilic stator, and subunit interactions dictate alternate-site catalysis. To explore the relevance of these interactions for catalysis we use hydrostatic pressure to induce conformational changes and/or subunit dissociation, and the resulting changes in the ... 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
Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.
AuthorsBlackman MJ, Corrie JE, Croney JC, Kelly G, Eccleston JF, Jameson DM
JournalBiochemistry
PubMed ID12356327
'Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine ... More
Molecular recognition by calmodulin: pressure-induced reorganization of a novel calmodulin-peptide complex.
AuthorsEhrhardt MR, Erijman L, Weber G, Wand AJ
JournalBiochemistry
PubMed ID8634291
'The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by fluorescence spectroscopy. The 1:1 complex (12 microM) formed between apoCaM and the Neurop peptide is extremely sensitive to salt and is half dissociated in less than ... More
Substructure and accessory proteins in scallop myosin filaments.
AuthorsVibert P, Castellani L
JournalJ Cell Biol
PubMed ID2760107
'Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. ... More
Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.
AuthorsGan WB, Bishop DL, Turney SG, Lichtman JW
JournalJ Neurosci Methods
PubMed ID10598860
'We describe a method for in vivo confocal fluorescence imaging of synaptic terminals and subsequent electron microscopic reconstructions of the same terminals. By iontophoretically applying lipophilic dye to nerve terminals at a single neuromuscular junction with a sharp microelectrode in living neonatal mice, we were able to quickly label other ... More
Deterministic pressure dissociation and unfolding of triose phosphate isomerase: persistent heterogeneity of a protein dimer.
AuthorsRietveld AW, Ferreira ST
JournalBiochemistry
PubMed ID8672474
'Subunit dissociation and unfolding of dimeric rabbit muscle triose phosphate isomerase (TIM) induced by hydrostatic pressure were investigated. Changes in fluorescence emission of TIM (both intrinsic and of covalently attached probes) indicated that pressure ranging from 1 bar to 3.5 kbar promoted subunit dissociation and unfolding. Instrinsic fluorescence changes upon ... More
Conformational state of ovalbumin at acidic pH as evaluated by a novel approach utilizing intrachain sulfhydryl-mixed disulfide exchange reactions.
AuthorsTatsumi E, Yoshimatsu D, Hirose M
JournalBiochemistry
PubMed ID9724549
'Ovalbumin contains four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) and one cystine disulfide (Cys73-Cys120). A highly reactive aromatic disulfide, 2,2'-dipyridyl disulfide, reacts specifically with Cys367 of ovalbumin at pH 2.2 generating a mixed disulfide protein derivative [Tatsumi, E., and Hirose, M. (1997) J. Biochem. 122, 300-308]. The mode of ... More
Localization of the phalloidin and nucleotide-binding sites on actin.
AuthorsBarden JA, Miki M, Hambly BD, Dos Remedios CG
JournalEur J Biochem
PubMed ID3830158
'Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy-transfer spectroscopy. ... More
Protease-sensitive regions in myosin subfragment 1.
AuthorsApplegate D, Reisler E
JournalProc Natl Acad Sci U S A
PubMed ID6359163
'Proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, thermolysin, and Staphylococcus aureus protease reveal that the two trypsin-sensitive regions in S-1 have broad protease susceptibility. The cleavage of S-1 by these enzymes yields products that correspond within 1-2 kilodaltons (kDa) to the 25-, 50-, and 20-kDa fragments ... More
Fluorescence energy transfer distance measurements. The hydrophobic helical hairpin of colicin A in the membrane bound state.
AuthorsLakey JH, Duché D, González-Mañas JM, Baty D, Pattus F
JournalJ Mol Biol
PubMed ID7683055
'The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this study fluorescence resonance energy ... More
Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.
AuthorsBorovikov YS, Moraczewska J, Khoroshev MI, Strzelecka-Golaszewska H
JournalBiochim Biophys Acta
PubMed ID10719182
'Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers ... More
Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin.
AuthorsMorita J, Takashi R, Ikebe M
JournalBiochemistry
PubMed ID1832559
'The 20,000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 degrees C. The light chain was almost completely exchanged within 30 min under the above conditions. The exchange was markedly inhibited either below ... More
19F NMR study of the myosin and tropomyosin binding sites on actin.
AuthorsBarden JA, Phillips L
JournalBiochemistry
PubMed ID2108725
'Actin was labeled with pentafluorophenyl isothiocyanate at Lys-61. The label was sufficiently small not to affect the rate or extent of actin polymerization unlike the much larger fluorescein 5-isothiocyanate which completely inhibits actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. Furthermore, the label resonances in the 376.3-MHz ... More
Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.
AuthorsYates SP, Merrill AR
JournalBiochem J
PubMed ID14733615
'Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA ... More
Activation of regulated actin by SH1-modified myosin subfragment 1.
AuthorsBobkov AA, Bobkova EA, Homsher E, Reisler E
JournalBiochemistry
PubMed ID9201914
'The reactive SH1 (Cys-707) group of the myosin subfragment 1 (S1) has been used frequently as an attachment site for fluorescent and spin probes in solution and muscle fiber experiments. In this study we examined (i) the motor function of SH1 spin-labeled heavy meromyosin (HMM) in the in vitro motility ... More
Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I.
'Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin I(129-166) complex demonstrated that cardiac troponin I(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and (15)N transverse relaxation rates for intact ... More
Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits.
AuthorsLeavis PC, Gowell E, Tao T
JournalBiochemistry
PubMed ID6487595
'Fluorescence lifetime and acrylamide quenching studies were carried out to characterize the interactions between the subunits of troponin under various conditions of metal ion binding. Troponin C was labeled at Cys-98 with N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine. In the presence of Ca2+, the fluorescence decay of labeled troponin C (TnC*) was monoexponential, lifetime tau ... More
Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase.
AuthorsReyes AM, Ludwig HC, Yañez AJ, Rodríguez PH, Slebe JC
JournalBiochemistry
PubMed ID12795590
'The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving ... More
The Par-3 NTD adopts a PB1-like structure required for Par-3 oligomerization and membrane localization.
AuthorsFeng W, Wu H, Chan LN, Zhang M
JournalEMBO J
PubMed ID17476308
'The evolutionarily conserved Par-3/Par-6/aPKC complex is essential for the establishment and maintenance of polarity of a wide range of cells. Both Par-3 and Par-6 are PDZ domain containing scaffold proteins capable of binding to polarity regulatory proteins. In addition to three PDZ domains, Par-3 also contains a conserved N-terminal oligomerization ... More
Use of fluorescence resonance energy transfer to estimate intramolecular distances in the Msx-1 homeodomain.
AuthorsIsaac VE, Patel L, Curran T, Abate-Shen C
JournalBiochemistry
PubMed ID7578143
'We have utilized fluorescence resonance energy transfer (FRET) to investigate the spatial proximities of segments in the Msx-1 homeodomain (Msx). This strategy makes use of a single, invariant tryptophan (Trp-48) in helix III as the donor for FRET. The acceptor molecule, 5-[[[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (AEDANS), was incorporated into Msx at positions ... More
Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.
AuthorsAlexandrov K, Simon I, Yurchenko V, Iakovenko A, Rostkova E, Scheidig AJ, Goody RS
JournalEur J Biochem
PubMed ID10491170
'Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein ... More
Dual labeling of a binding protein allows for specific fluorescence detection of native protein.
AuthorsKarlström A, Nygren PA
JournalAnal Biochem
PubMed ID11476541
'Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from ... More
Cysteine 532 and cysteine 545 are the N-ethylmaleimide-reactive residues of the Neurospora plasma membrane H+-ATPase.
AuthorsPardo JP, Slayman CW
JournalJ Biol Chem
PubMed ID2524486
'Previous studies from this laboratory (Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226; Davenport, J. W., and Slayman, C. W. (1988) J. Biol. Chem. 263, 16007-16013) have used the sulfhydryl reagent N-ethylmaleimide (NEM) to define two sites on the Neurospora plasma membrane H+-ATPase: a "fast" ... More
Intradomain distances in the regulatory domain of the myosin head in prepower and postpower stroke states: fluorescence energy transfer.
AuthorsPalm T, Sale K, Brown L, Li H, Hambly B, Fajer PG
JournalBiochemistry
PubMed ID10529172
'The relative movement of the catalytic and regulatory domains of the myosin head (S1) is likely to be the force generating conformational change in the energy transduction of muscle [Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science ... More
Fluorescence labeling of an aminoacyl-tRNA at the 3'-end and its interaction with elongation factor Tu.GTP.
AuthorsJoshi RL, Faulhammer HG, Haenni AL, Sprinzl M
JournalFEBS Lett
PubMed ID3536575
'A new approach for the fluorescence labeling of an aminoacyl-tRNA at the 3''-end is applied to study its interaction with bacterial elongation factor Tu (EF-Tu) and GTP at equilibrium. The penultimate cytidine residue in yeast tRNATyr-C-C-A was replaced by 2-thiocytidine (s2C). The resulting tRNATyr-C-s2C-A was aminoacylated and then alkylated at ... More
Denatured state of ovalbumin in high concentrations of urea as evaluated by disulfide rearrangement analysis.
AuthorsTatsumi E, Takahashi N, Hirose M
JournalJ Biol Chem
PubMed ID7961742
'To investigate the highly denatured state of ovalbumin (molecular mass of 42.7 kDa, four cysteine sulfhydryls and one cystine disulfide) using the disulfide rearrangement approach, we established the peptide-mapping procedure using a cysteine-labeling technique with a fluorescent dye that allows the quantitative analyses for the disulfide-involved half-cystines. Ovalbumin denatured at ... More
Outer membrane phospholipase A is dimeric in phospholipid bilayers: a cross-linking and fluorescence resonance energy transfer study.
AuthorsUbarretxena-Belandia I, Hozeman L, van der Brink-van der Laan E, Pap EH, Egmond MR, Verheij HM, Dekker N
JournalBiochemistry
PubMed ID10353852
'In the cell, the activity of outer membrane phospholipase A (OMPLA) is strictly regulated to prevent uncontrolled breakdown of the membrane lipids. Previously, it has been shown that the enzymatic activity is modulated by reversible dimerization. The current studies were carried out to define the oligomeric state of OMPLA in ... More
Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy.
AuthorsGryczynski I, Wiczk W, Inesi G, Squier T, Lakowicz JR
JournalBiochemistry
PubMed ID2525924
'We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ ... More
Effect of denervation, reinnervation and hypertrophy on the state of actin filaments in skeletal muscle fibres.
AuthorsSzczepanowska J, Borovikov YS, Jakubiec-Puka A
JournalEur J Cell Biol
PubMed ID3622526
'The conformational state of actin filaments was studied in the rat soleus muscle atrophying after denervation, recovering following reinnervation, hypertrophying following tenotomy of synergists and in intact muscle. Intrinsic (tryptophan residues of F-actin) and extrinsic (rhodamine-phalloidin or 1,5-IAEDANS attached to F-actin) polarized fluorescence was measured. In parallel, the influence of ... More
Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.
AuthorsTao T, Gong BJ, Leavis PC
JournalScience
PubMed ID2138356
'The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between ... More
The myosin head can bind two actin monomers.
AuthorsAndreev OA, Borejdo J
JournalBiochem Biophys Res Commun
PubMed ID2043120
'Force impulse is thought to be generated in muscle when myosin head (S-1), while weakly bound to actin filament, undergoes orientational change to form a strong (rigor) bond with actin. There is ample evidence that this bond involves interaction of 1 myosin head with 1 actin monomer. However, X-ray diffraction ... More
Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase beta 2 subunit.
AuthorsMurry-Brelier A, Goldberg ME
JournalBiochemistry
PubMed ID2462907
'A reversible acid-denaturation process of the beta 2 subunit of Escherichia coli tryptophan synthase has been set up. The acid-denatured state has been physically characterized: though not in a random-coiled conformation, it is extensively denatured. The renaturation of this denatured state of beta 2 has been observed in a stopped-flow ... More
Evidence for the association between two myosin heads in rigor acto-smooth muscle heavy meromyosin.
AuthorsOnishi H, Maita T, Matsuda G, Fujiwara K
JournalBiochemistry
PubMed ID2524210
'The rigor complexes that formed between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), in which the heavy chains had been cleaved with trypsin into 24K, 50K, and 68K fragments, were examined by using the zero-length chemical cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Two cross-linked products of approximate Mr 115K and ... More
Fluorescent labeling of cysteinyl residues. Application to extensive primary structure analysis of proteins on a microscale.
AuthorsGorman JJ, Corino GL, Mitchell SJ
JournalEur J Biochem
PubMed ID3311742
'The specificity and efficiency of fluorescent labeling of proteins by reduction and subsequent alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) [J.J. Gorman, (1987) Anal. Biochem. 160, 376-387] has been investigated. Proteins studied include porcine insulin, chicken ovalbumin and bovine serum albumin. Amino acid analysis of the B-chain derivative of insulin revealed quantitative ... More
Configurations of the N-terminal amphipathic domain of the membrane-bound M13 major coat protein.
AuthorsMeijer AB, Spruijt RB, Wolfs CJ, Hemminga MA
JournalBiochemistry
PubMed ID11305925
'The M13 major coat protein has been extensively studied in detergent-based and phospholipid model systems to elucidate its structure. This resulted in an L-shaped model structure of the protein in membranes. An amphipathic alpha-helical N-terminal arm, which is parallel to the surface of the membrane, is connected via a flexible ... More
Preparation of fluorescently labelled hybrid histone octamers.
AuthorsLindsey GG, Thompson P
JournalBiochim Biophys Acta
PubMed ID2357469
'Labelling hybrid histone octamers (the Cys variant of histone H4 replaced histone H4 in the chicken erythrocyte octamer) with the fluorescent probe 5-(2(iodoacetyl)aminoethyl)aminonapthalene- 1-sulfonic acid, IAEDANS, resulted in significant non-specific incorporation of label. Fluorescently labelled hybrid histone octamers were prepared by reconstitution methodology after labelling the isolated histone Cys-H4 and ... More
Actin-dystrophin interface.
AuthorsFabbrizio E, Bonet-Kerrache A, Leger JJ, Mornet D
JournalBiochemistry
PubMed ID8399191
'Dystrophin, an elongated cytoskeletal molecule which is deficient in Duchenne muscular disease, contains an actin-binding domain in its N-terminal portion. We show that this part interacted with actin in the native molecule. By molecular biology techniques, four recombinant proteins were expressed in Escherichia coli using the pMAL vector which allowed ... More
Calcium-dependent structural coupling between opposing globular domains of calmodulin involves the central helix.
AuthorsSun H, Yin D, Squier TC
JournalBiochemistry
PubMed ID10493794
'We have used fluorescence spectroscopy to investigate the average structure and extent of conformational heterogeneity associated with the central helix in calmodulin (CaM), a sequence that contributes to calcium binding sites 2 and 3 and connects the amino- and carboxyl-terminal globular domains. Using site-directed mutagenesis, a double mutant was constructed ... More